0711 月/24

KMD Bioscience-Crispr Cas9 Library Screening

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CRISPR-Cas9 library screening is a high-throughput genetic screening technique used to investigate the function of genes across the entire genome or within a subset of genes. This approach leverages the CRISPR-Cas9 system to create targeted mutations or gene knockouts in cells, allowing researchers to assess the impact of each gene on a particular phenotype, such as cell survival, proliferation, or response to a drug.

Overview of CRISPR-Cas9 Library Screening

CRISPR-Cas9 library screening involves delivering a library of guide RNAs (gRNAs) that target numerous genes in the genome to a population of cells. Each gRNA directs the Cas9 enzyme to cut a specific gene, which either disrupts or modifies the gene function. This process is used to study the role of specific genes in a biological process by observing the outcome of the gene disruptions.

There are two main types of CRISPR library screens:

Loss-of-Function (Knockout) Screens: Aim to identify genes that are critical for a particular function by knocking them out and observing the resulting phenotype.

Gain-of-Function or Activation Screens: CRISPRa (CRISPR activation) can be used to activate genes and investigate their overexpression effects.

 Steps in CRISPR-Cas9 Library Screening

Designing the gRNA Library

The gRNA library consists of thousands to tens of thousands of gRNAs, each designed to target a specific gene.

The libraries can target either the entire genome (genome-wide screens) or specific sets of genes (focused screens). Each gene is typically targeted by multiple gRNAs to ensure robust disruption and accurate results.

Libraries are typically synthesized as pooled oligonucleotides.

Transduction into Cells

The CRISPR gRNA library is delivered to cells using lentiviral vectors. These vectors enable stable integration of gRNAs into the genomes of the target cells.

Cells are also transduced with Cas9 if they don’t already express it. Some screening systems use cell lines that stably express Cas9, making it easier to conduct screens.

The number of cells transduced is important to maintain complexity. The number of cells should exceed the number of gRNAs in the library to ensure that each gRNA is represented in multiple cells.

Selection of Cells

After the gRNAs and Cas9 are introduced, the cells are allowed to proliferate for several days, giving time for the CRISPR-Cas9 system to create gene knockouts.

Cells are then subjected to a selection process, which can be based on a particular phenotype (e.g., resistance to a drug, survival under specific stress, or enhanced proliferation).

This step isolates the cells that have undergone genetic changes leading to the desired phenotype.

Next-Generation Sequencing (NGS)

After selection, the gRNAs present in the surviving cells are identified by extracting genomic DNA and sequencing the gRNA regions.

NGS provides a readout of which gRNAs were enriched (suggesting that the targeted genes are involved in promoting the selected phenotype) or depleted (indicating that the targeted genes are essential for cell viability under the given conditions).

Data Analysis

The sequencing data is analyzed to determine which gRNAs (and thus which genes) are associated with the desired phenotype.

Enrichment or depletion of certain gRNAs is compared to a control population (untreated or non-selected cells), and statistical methods are used to identify significant hits.

Hits from the screen can then be validated through further experimental testing, such as individual gene knockouts or knockdowns.

 Applications of CRISPR-Cas9 Library Screening

Functional Genomics

CRISPR library screens are used to study the function of genes across the entire genome, allowing researchers to assign roles to previously uncharacterized genes.

Genome-wide screens can reveal essential genes for fundamental processes like cell division, apoptosis, or differentiation.

Cancer Research

CRISPR screening helps identify genes that are critical for cancer cell survival or proliferation, making them potential therapeutic targets.

For example, screens can reveal genes that cancer cells rely on under specific stress conditions, such as treatment with chemotherapy drugs.

CRISPR screens are also useful for studying genes involved in resistance to cancer therapies, helping to develop strategies to overcome resistance.

Drug Target Identification

By knocking out genes, researchers can identify which genes are essential for the efficacy of certain drugs, revealing new drug targets or helping to repurpose existing drugs for new uses.

CRISPR screens can be performed in the presence of drug candidates to identify genes that modulate the drug’s activity, such as resistance mechanisms or synergistic interactions.

Synthetic Lethality Screens

In synthetic lethality screens, CRISPR is used to knock out genes in cells that already have certain mutations, with the goal of identifying gene pairs where the loss of both genes is lethal to the cell.

This approach is valuable for finding vulnerabilities in cancer cells, where knocking out specific genes in combination with cancer-related mutations can selectively kill the tumor cells.

Immunology

CRISPR screens can be used to identify genes involved in immune cell function, such as T cell activation or cytokine production.

This is useful for understanding immune regulation and identifying targets for immunotherapy.

Neuroscience

In neuroscience, CRISPR screens can help discover genes that play critical roles in neuronal function, development, and diseases like neurodegeneration.

 Types of CRISPR-Cas9 Library Screens

Positive Selection Screens

In these screens, cells with beneficial mutations (e.g., those that confer drug resistance or enhanced proliferation) are selected and enriched over time.

gRNAs that are enriched indicate the genes whose disruption promotes the selected phenotype.

Negative Selection Screens

These screens identify genes that are essential for survival under specific conditions. Cells with deleterious mutations (e.g., loss of an essential gene) will be depleted.

gRNAs that are depleted indicate the genes that are important for cell survival or fitness.

 Advantages of CRISPR-Cas9 Library Screening

High Throughput: CRISPR-Cas9 library screening allows researchers to assess the function of thousands of genes in a single experiment, providing a powerful way to perform large-scale genetic screens.

Precision: CRISPR knockouts are highly specific and permanent, offering a more precise approach compared to RNA interference (RNAi) knockdown techniques, which often result in incomplete or transient gene suppression.

Versatility: CRISPR libraries can be designed to knock out, activate, or repress genes, making this approach suitable for a wide range of applications.

 Challenges and Limitations

Off-Target Effects: Although CRISPR is highly specific, off-target activity can still occur, leading to unintended mutations.

Incomplete Knockout: Not all gRNAs will efficiently knock out their target genes, and some gRNAs may only partially reduce gene function.

Library Complexity: Proper representation of all gRNAs in a complex library requires careful experimental design, including maintaining high cell numbers to ensure that every gRNA is represented in multiple cells.

Data Interpretation: Identifying the most relevant hits from a large dataset can be challenging and requires robust statistical analysis. Follow-up experiments are often needed to validate key findings.

 Conclusion

CRISPR-Cas9 library screening is a powerful tool for large-scale genetic exploration, enabling the identification of genes involved in various biological processes, disease mechanisms, and drug responses. It has revolutionized functional genomics, cancer research, and drug discovery, and continues to drive new discoveries in biology. By allowing researchers to systematically disrupt genes and observe the consequences, this technique provides unparalleled insight into gene function and potential therapeutic targets.

0611 月/24

KMD Bioscience-Stable Cell Line Generation

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Stable cell line generation refers to creating cells that have been genetically modified to continuously express a gene or sequence of interest over many generations. This technique is widely used in biological research, biotechnology, and pharmaceutical development for the production of proteins, gene function studies, and drug screening.

 Process of Stable Cell Line Generation

The key steps to generating a stable cell line include transfecting cells with a gene of interest, selecting cells that have successfully integrated the gene into their genome, and ensuring that these modifications are passed on to future generations. Below are the steps involved:

Selection of a Parental Cell Line

A suitable cell line (e.g., HEK293, CHO, or HeLa cells) is chosen based on the experimental needs. The cell line should be easily transfected, express the gene of interest effectively, and grow well in culture.

Transfection

The gene of interest is introduced into the cells using a transfection method. The gene is often cloned into a plasmid vector that also contains a selection marker (like antibiotic resistance genes) to identify cells that have successfully taken up the plasmid.

Common transfection methods include

Lipofection: Using lipid-based reagents to encapsulate DNA for delivery into cells.

Electroporation: Using electrical pulses to create pores in the cell membrane, allowing DNA to enter.

Viral transduction: Using viral vectors like lentivirus or retrovirus to deliver the gene of interest.

Selection of Stable Integrants

After transfection, not all cells will integrate the gene into their genome. To identify the stable integrants (cells that have incorporated the gene into their chromosomal DNA), a selection agent (e.g., antibiotic like G418, puromycin, or hygromycin) is added to the culture medium.

Only the cells that have successfully integrated the plasmid containing the antibiotic resistance gene will survive this selection process.

Colony Isolation: After selection, surviving cells are isolated, often using techniques like clonal dilution or single-cell sorting (e.g., fluorescence-activated cell sorting, FACS). Individual colonies are then expanded into larger cultures.

Screening and Characterization

Once colonies of potentially stable cell lines are established, they need to be screened to confirm the successful integration and expression of the gene of interest. Screening methods include:

PCR: To verify the presence of the gene in the genome.

  Western blotting or ELISA: To confirm protein expression.

Flow cytometry: For cell surface or intracellular protein expression.

Additionally, the stability of gene expression is assessed by culturing the cells over many generations to ensure consistent expression over time.

Expansion of Stable Cell Lines

Once the stable clones have been identified and characterized, they are expanded to create large populations of cells that can be used in downstream experiments.

The cell lines can be stored by cryopreservation to ensure long-term availability.

Key Components of Stable Cell Line Generation

1. Selection Markers: These are used to ensure that only cells which have integrated the gene of interest survive. Common selection markers include:

Antibiotic resistance genes: (e.g., neomycin/G418 resistance, puromycin resistance, hygromycin resistance).

Fluorescent markers: GFP (green fluorescent protein) can be co-expressed to visually identify cells expressing the gene of interest.

2. Promoter Choice: The promoter used in the plasmid vector controls the expression of the gene of interest. Common promoters include:

CMV (cytomegalovirus promoter): A strong, ubiquitous promoter often used for high expression.

EF1α (elongation factor-1 alpha promoter): Another widely used promoter for consistent gene expression.

Inducible Promoters: For conditional expression, promoters like TET-on/off systems can be used, allowing for temporal control over gene expression.

3. Integration Methods:

Random Integration: Traditional methods rely on random integration of the plasmid into the genome. This can lead to variable expression depending on the site of integration.

Site-Specific Integration (CRISPR/Cas9 or Recombinase Systems): Modern techniques use CRISPR/Cas9 or recombinases like Cre-Lox or Flp-In systems to integrate the gene of interest into specific, well-characterized loci, reducing variability and improving consistency of gene expression.

 Applications of Stable Cell Lines

Protein Production: Stable cell lines are commonly used for the large-scale production of recombinant proteins, such as therapeutic antibodies, vaccines, or enzymes.

Example: Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry for the production of monoclonal antibodies.

Gene Function Studies: Stable cell lines allow researchers to study the effects of overexpression or knockdown of specific genes over long periods.

Example: Researchers use stable cell lines to express mutant genes to study their role in diseases like cancer or neurodegeneration.

Drug Screening and Development: Pharmaceutical companies use stable cell lines expressing a target protein to screen potential drug candidates.

Example: Cells stably expressing a receptor of interest are used to identify small molecules that inhibit or activate the receptor.

CRISPR/Cas9 Studies: Stable cell lines expressing Cas9 or a guide RNA (gRNA) are used for genome editing experiments, enabling researchers to knock out or modify genes over time.

 Challenges and Considerations

Time-Consuming: The process of generating stable cell lines can take several weeks to months, depending on the cell type and the complexity of the modifications.

Clonal Variability: Since random integration is common, each clone may show different levels of gene expression. Screening multiple clones is necessary to find one with the desired expression profile.

Gene Silencing: In some cases, even if the gene of interest is integrated, it may get silenced over time, particularly if integrated into heterochromatin regions. Site-specific integration methods can help mitigate this risk.

Off-Target Effects: If CRISPR or other gene-editing tools are used, off-target effects should be considered, which may affect the stability and behavior of the cell line.

Conclusion

Stable cell line generation is an essential technique in molecular biology and biotechnology, enabling the long-term and consistent expression of genes for research and therapeutic purposes. The choice of transfection method, selection marker, and screening process are critical to successfully creating cell lines with the desired characteristics. As technology advances, more precise and efficient methods are being developed to improve the generation and reliability of stable cell lines, which are indispensable in fields like drug discovery, gene therapy, and biomanufacturing.

3110 月/24

KMD Bioscience-Cas9 Gene Editing

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Cas9 gene editing, commonly referred to as CRISPR-Cas9 gene editing, is a revolutionary technology that enables precise modifications of DNA in living organisms. It is based on a natural defense mechanism found in bacteria, where the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, along with the Cas9 protein, targets and cuts foreign viral DNA. Scientists have harnessed this system to edit genes in a wide variety of organisms, including humans.

Key Components of CRISPR-Cas9

Cas9 Protein

Cas9 (CRISPR-associated protein 9) is an endonuclease enzyme that can cut double-stranded DNA at a specific location. It is directed to the DNA target by a guide RNA (gRNA).

Guide RNA (gRNA)

The gRNA is a synthetic RNA molecule that contains two parts:

A CRISPR RNA (crRNA): This part is complementary to the target DNA sequence and directs the Cas9 protein to the specific location in the genome.

A trans-activating crRNA (tracrRNA): This part binds to the Cas9 protein and activates it for DNA cleavage.

Together, these components form a single guide RNA (sgRNA), which directs Cas9 to the precise DNA sequence to be edited.

PAM Sequence

The Protospacer Adjacent Motif (PAM) is a short DNA sequence (typically “NGG”) located next to the target site. Cas9 requires the PAM sequence to bind and cut the DNA at the correct location.

How CRISPR-Cas9 Gene Editing Works

Design of Guide RNA

Researchers design a guide RNA sequence that is complementary to the target DNA region they want to edit. The target sequence must be adjacent to a PAM sequence to be recognized by Cas9.

Introduction into Cells

The Cas9 protein and guide RNA are introduced into the target cells via methods such as plasmids, viral vectors, or ribonucleoprotein complexes (pre-assembled Cas9 and gRNA).

DNA Binding and Cutting

Once inside the cell, the gRNA directs the Cas9 protein to the target DNA sequence. Cas9 binds to the DNA and cuts both strands at the specific site.

DNA Repair

After the DNA is cut, the cell’s natural DNA repair mechanisms are activated. There are two main repair pathways:

Non-Homologous End Joining (NHEJ): This is an error-prone repair process that often introduces small insertions or deletions (indels) at the cut site, leading to gene disruption or knockout.

Homology-Directed Repair (HDR): If a donor DNA template is provided along with Cas9, the cell can use this template to precisely repair the break, allowing for specific changes such as gene insertion, correction, or replacement.

Applications of CRISPR-Cas9 Gene Editing

Gene Knockout

Knocking out a gene involves creating a break in the DNA, followed by NHEJ, which introduces indels that disrupt the gene’s function. This is useful for studying the function of specific genes by observing the effects of their loss.

Gene Correction or Replacement

Using HDR with a supplied template DNA, CRISPR-Cas9 can make precise edits to correct mutations, introduce desired mutations, or replace faulty genes. This has potential for gene therapy in genetic disorders.

Functional Genomics

CRISPR-Cas9 is used in large-scale screens to study the function of genes across the genome. By knocking out thousands of genes in parallel, researchers can identify genes involved in specific biological processes or diseases.

Disease Modeling

CRISPR-Cas9 is used to create models of human diseases in animals or cell cultures by introducing specific mutations that mimic the disease condition. These models help in understanding the disease mechanisms and in drug testing.

Gene Therapy

CRISPR-Cas9 holds great promise for gene therapy by allowing precise correction of genetic mutations in patients. For example, it has been used in preclinical and clinical trials to treat conditions like sickle cell disease, beta-thalassemia, and certain forms of blindness.

Agriculture

In plant and animal breeding, CRISPR-Cas9 is used to introduce beneficial traits, such as disease resistance, drought tolerance, or enhanced nutritional content, into crops and livestock.

Cancer Research

CRISPR-Cas9 is being used to edit cancer-related genes in cell lines and animal models, helping to identify new targets for cancer therapies and understand tumor biology.

Advantages of CRISPR-Cas9

Precision: CRISPR-Cas9 can target specific regions in the genome with high accuracy, enabling precise edits.

Efficiency: Compared to earlier gene-editing techniques like TALENs and zinc-finger nucleases, CRISPR-Cas9 is simpler, faster, and more cost-effective.

Versatility: CRISPR-Cas9 can be used for a wide range of applications, from gene knockout to complex gene corrections and insertions.

Multiplexing: Multiple genes can be edited simultaneously by introducing multiple gRNAs, allowing for large-scale genetic screens and studies.

 Limitations and Challenges

Off-Target Effects

Sometimes, Cas9 can bind to DNA sequences that are similar but not identical to the target sequence, leading to off-target cuts. This can cause unwanted mutations in other parts of the genome.

Efficiency of HDR

HDR occurs less frequently than NHEJ, which makes precise editing (such as gene correction or insertion) less efficient. This is a challenge for gene therapy applications where precise edits are crucial.

Delivery

Efficiently delivering CRISPR-Cas9 components into the target cells, especially in vivo (within living organisms), remains a challenge. Delivery methods such as viral vectors or nanoparticles are being developed to improve this.

Ethical Concerns

The ability to edit the genome raises ethical questions, especially regarding germline editing (editing sperm, eggs, or embryos), which could lead to heritable changes. Concerns about unintended consequences and the potential for “designer babies” have led to calls for regulations and guidelines.

 Recent Advances

CRISPR-Cas9 Variants

Base Editors: Instead of making double-strand breaks, base editors can convert one DNA base to another (e.g., C to T or A to G) without causing a double-stranded break. This allows for more precise editing.

Prime Editing: A newer form of CRISPR that allows for more flexible and precise changes to the DNA without relying on double-strand breaks or donor templates.

CRISPRi and CRISPRa

CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) are techniques that use a “dead” version of Cas9 (dCas9) that can no longer cut DNA but can still bind to specific sequences. By recruiting repressor or activator proteins, CRISPRi can be used to silence genes, and CRISPRa can be used to increase gene expression.

 Summary

CRISPR-Cas9 gene editing is a groundbreaking tool that has transformed genetic research, offering precise, efficient, and versatile genome modification capabilities. Its applications range from basic research to therapeutic development, with the potential to treat genetic disorders, improve agriculture, and advance personalized medicine. However, challenges such as off-target effects and ethical considerations must be addressed as the technology continues to develop.

3010 月/24

KMD Bioscience-Western Blot Steps

Protein Engineering

Here is a detailed breakdown of the steps involved in performing a Western blot:

Western Blot Steps

Sample Preparation

Lysis of Cells or Tissues:

Lyse the cells or tissues to extract proteins using an appropriate lysis buffer (e.g., RIPA buffer) containing protease and phosphatase inhibitors to prevent protein degradation.

Protein Quantification:

Quantify the total protein concentration using a protein quantification assay like the BCA or Bradford assay.

Denaturation:

Mix equal amounts of protein with Laemmli sample buffer (contains SDS, β-mercaptoethanol or DTT, glycerol, and a tracking dye).

Boil the samples at 95°C for 5-10 minutes to denature the proteins and reduce disulfide bonds.

SDS-PAGE (Gel Electrophoresis)

Preparation of Polyacrylamide Gel:

Use a pre-cast gel or prepare your own using a polyacrylamide gel (usually a stacking gel of 4% and a resolving gel of 6-15%, depending on protein size).

Loading the Gel:

Load equal amounts of protein (typically 20-40 µg per well) along with a protein molecular weight marker (ladder) to determine protein size.

Running the Gel:

Run the gel in SDS-PAGE running buffer at 100-150 V for 1-2 hours. Proteins will separate by molecular weight, with smaller proteins moving faster through the gel.

Protein Transfer to Membrane

Prepare the Transfer Sandwich:

After electrophoresis, the gel is placed in a transfer “sandwich” with a membrane (either nitrocellulose or PVDF) in between filter papers and sponges, ensuring no air bubbles between the gel and membrane.

Transfer Proteins:

Transfer the proteins from the gel onto the membrane using electrophoretic transfer.

Transfer can be done via wet transfer (90 minutes at 100 V or overnight at low voltage at 4°C) or semi-dry transfer (10-30 minutes).

Check Protein Transfer:

Optional: Stain the membrane with Ponceau S to visualize protein transfer.

Blocking the Membrane

Blocking Buffer:

Block non-specific sites on the membrane by incubating it in blocking buffer (commonly 5% non-fat milk or BSA in TBST) for 1 hour at room temperature or overnight at 4°C.

Blocking prevents the antibodies from binding non-specifically to the membrane and reduces background noise.

Primary Antibody Incubation

Add Primary Antibody:

Incubate the membrane with the primary antibody specific to your target protein. The antibody should be diluted in blocking buffer or TBST (typical dilutions range from 1:500 to 1:5000).

Incubation Time:

Incubate for 1-2 hours at room temperature or overnight at 4°C with gentle shaking.

Washing:

Wash the membrane 3-5 times with TBST (Tris-buffered saline with Tween-20) to remove unbound primary antibody.

Secondary Antibody Incubation

Add Secondary Antibody:

Incubate the membrane with a secondary antibody that is conjugated to an enzyme, typically horseradish peroxidase (HRP) or alkaline phosphatase (AP). This antibody binds to the primary antibody.

Dilute the secondary antibody in blocking buffer or TBST (typically 1:5000 to 1:20,000).

Incubation Time:

Incubate for 1-2 hours at room temperature with gentle shaking.

Washing:

Wash the membrane 3-5 times in TBST to remove unbound secondary antibody.

Protein Detection

Substrate Application:

Add a substrate specific to the enzyme conjugated to the secondary antibody. For HRP, add a chemiluminescent substrate like ECL (enhanced chemiluminescence).

Signal Detection:

Detect the emitted signal using X-ray film or a digital imaging system. The chemiluminescence reaction will produce visible bands at the location of the target protein, which can then be visualized and quantified.

Data Analysis

Quantification:

Compare the intensity of the protein bands across different samples. Normalize the intensity of the target protein to a loading control (such as β-actin, GAPDH, or tubulin) to account for any variations in loading amounts.

Densitometry:

Use imaging software to measure the intensity of the protein bands and calculate relative expression levels.

Controls in Western Blotting

Loading Control

A housekeeping protein such as β-actin, GAPDH, or tubulin is used to ensure equal protein loading across samples.

Positive Control

A known sample that expresses the target protein, ensuring the antibodies and conditions work.

Negative Control

A sample lacking the target protein to confirm the specificity of the antibody.

 Troubleshooting

Weak Signal

Increase primary or secondary antibody concentrations, or extend the exposure time.

High Background

Ensure adequate washing and use a more stringent blocking buffer. Consider diluting the antibody or reducing the exposure time.

Non-Specific Bands

Use a more specific primary antibody and optimize blocking/washing steps.

 Summary

The Western blot procedure provides a powerful and sensitive method for detecting specific proteins in a sample, offering insights into protein expression levels, molecular weight, and post-translational modifications. By following these steps and optimizing each stage, researchers can achieve accurate and reproducible results.

2910 月/24

KMD Bioscience-Flow Cytometry Assay

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A flow cytometry assay is a method used to measure various physical and chemical characteristics of cells or particles using flow cytometry. Flow cytometry assays are highly versatile and allow for the simultaneous analysis of multiple parameters, such as cell size, granularity, and the expression of specific surface or intracellular markers. They are widely used in research, diagnostics, and clinical applications, particularly in immunology, cancer research, and drug development.

Steps for Developing a Flow Cytometry Assay

Define the Objective

The first step in designing a flow cytometry assay is to clearly define the purpose of the experiment. This could include identifying specific cell populations (e.g., immune cell subsets), measuring the expression of specific proteins (e.g., surface receptors), or assessing cellular processes (e.g., apoptosis, cell cycle).

Selection of Fluorochrome-Conjugated Antibodies

Choose fluorochrome-conjugated antibodies that bind to specific cell surface or intracellular markers relevant to the target cells or molecules.

Ensure the fluorochromes have minimal spectral overlap, especially for multicolor assays.

Commonly used fluorochromes include FITC, PE, APC, and PerCP.

Ensure the antibodies are validated for flow cytometry and specific to the species and cell types being analyzed.

Sample Preparation

Prepare a single-cell suspension from the sample. Depending on the source, this could involve enzymatic digestion of tissue or simple preparation of blood or cell culture samples.

For adherent cells, detach them using trypsin or other dissociation enzymes.

Filter the sample through a mesh or filter to avoid clumping and ensure smooth flow through the cytometer.

Cell Staining

Surface Marker Staining: If detecting surface markers, incubate the cells with fluorochrome-conjugated antibodies specific to the proteins of interest. Typically, this is done for 20–30 minutes at 4°C in the dark to prevent photobleaching.

Intracellular Staining: For intracellular proteins (e.g., transcription factors, cytokines), fix and permeabilize the cells before staining. Common reagents for this purpose include paraformaldehyde (for fixation) and saponin or Triton X-100 (for permeabilization).

Include appropriate controls, such as:

Isotype controls to account for non-specific antibody binding.

Unstained controls to establish background fluorescence levels.

Single-stained controls for compensation when multiple fluorochromes are used.

Washing and Resuspension

After staining, wash the cells with a suitable buffer (e.g., phosphate-buffered saline (PBS) with 1-2% BSA) to remove unbound antibodies.

Resuspend the cells in flow cytometry buffer (PBS + 1% BSA) for acquisition. For live cells, a viability dye (e.g., propidium iodide, 7-AAD) can be used to exclude dead cells from analysis.

Flow Cytometer Setup

Set up the flow cytometer by configuring the appropriate lasers and detectors for the selected fluorochromes.

Compensation: In multicolor assays, spectral overlap between fluorophores needs to be corrected using compensation controls (single-stained samples for each fluorochrome).

Gating Strategy: Define gating regions based on forward scatter (FSC) and side scatter (SSC) to exclude debris and isolate the cell populations of interest (e.g., lymphocytes or monocytes).

Data Acquisition

Run the samples through the flow cytometer. As cells pass through the laser, the machine records data for each cell, including size (FSC), complexity (SSC), and fluorescence intensity for each fluorochrome.

Collect data from tens of thousands to millions of cells to ensure statistical accuracy and representation of all subpopulations.

Data Analysis

Gating: Identify and isolate specific cell populations based on fluorescence and scatter profiles. Gating strategies often involve:

FSC vs. SSC: To exclude debris and focus on the cell type of interest (e.g., lymphocytes, neutrophils).

Fluorescence intensity plots: To identify positive and negative populations for the markers of interest.

Histograms and dot plots are used to visualize data.

Quantify the percentage of cells expressing each marker or analyze the mean fluorescence intensity (MFI) to determine the expression level of specific proteins.

Controls and Validation

Use proper controls such as unstained cells, isotype controls, and fluorescence-minus-one (FMO) controls to accurately interpret the data and set gates.

For multicolor assays, ensure that compensation controls are accurate to prevent misinterpretation of overlapping fluorescence signals.

 Types of Flow Cytometry Assays:

Immunophenotyping

One of the most common uses of flow cytometry is to identify specific cell populations based on surface or intracellular markers. For example, CD4 and CD8 markers can be used to distinguish T cell subsets.

Important for diagnosing immune disorders (e.g., leukemia, lymphoma, HIV).

Cell Cycle Analysis

Flow cytometry can assess DNA content in cells to determine their cell cycle phase (G0/G1, S, or G2/M). DNA-binding dyes such as propidium iodide (PI) or DAPI are commonly used.

Useful for studying cell proliferation, cancer biology, and drug effects on the cell cycle.

Apoptosis Assays

Flow cytometry can detect early and late stages of apoptosis using markers such as Annexin V (for early apoptosis) and propidium iodide or 7-AAD (for necrosis or late apoptosis).

Essential for studying cell death in response to treatments or stress.

Cytokine Detection

Cytokines can be detected intracellularly after cell stimulation using fluorochrome-conjugated antibodies.

Intracellular cytokine staining (ICS) is widely used in immunology and vaccine research to measure cytokine production in response to antigenic stimulation.

Cell Proliferation Assays

Cell proliferation can be measured using dyes such as CFSE (carboxyfluorescein succinimidyl ester), which is diluted as cells divide.

This technique is useful for tracking cell division over time in both in vitro and in vivo studies.

Cell Viability Assays

Live/dead cell discrimination is essential in many experiments. Viability dyes such as 7-AAD, propidium iodide, and live/dead fixable stains are used to exclude dead cells from analysis.

 Key Considerations for Flow Cytometry Assay Development

Fluorochrome Selection

Choose fluorochromes that match the available lasers and detectors on your flow cytometer.

Avoid spectral overlap by using non-overlapping fluorochromes for multicolor experiments, and perform compensation to correct for any overlap.

Cell Density

Ensure proper cell density in the sample to avoid clogging the cytometer and to get optimal flow rates (typically 1 million cells/mL).

Instrument Calibration and Standardization

Regular calibration of the flow cytometer using calibration beads is essential for ensuring data accuracy and consistency across runs.

Controls

Use appropriate controls (e.g., isotype controls, single-stained controls) to set gates and accurately interpret the results.

Data Quality

Acquire enough events (cells) to ensure statistical reliability. For rare cell populations, collecting 100,000–1,000,000 events may be necessary.

 Applications of Flow Cytometry Assays

Immunology: Identification of immune cell subsets, cytokine production, and functional assays.

Cancer Research: Detection of cancer markers, minimal residual disease (MRD), and monitoring therapy responses.

Stem Cell Research: Identification and characterization of stem cells and progenitor cells.

Vaccine Development: Assessment of immune responses following vaccination.

Hematology: Diagnosis of blood disorders like leukemia and lymphoma.

Flow cytometry assays are crucial for studying cellular properties in real-time and offer high sensitivity, precision, and the ability to analyze multiple parameters simultaneously, making them invaluable in both research and clinical diagnostics.

2410 月/24

KMD Bioscience-Flow Cytometry Analysis

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Flow cytometry analysis is a powerful technique used to analyze the physical and chemical characteristics of cells or particles as they flow in a fluid stream through a laser beam. This technique is widely used in immunology, hematology, cancer research, and other fields of biology and medicine for cell counting, biomarker detection, and functional analysis of cells.

How Flow Cytometry Works:

Sample Preparation:

Cells or particles of interest are prepared in a single-cell suspension in a buffer solution. These could be blood cells, immune cells, or other types of cells derived from tissues, or even beads coated with specific molecules.

Cells are often stained with fluorescently-labeled antibodies or other markers that bind to specific proteins, such as surface antigens, intracellular proteins, or nucleic acids.

Flow Cytometer Components:

Fluidics System: The sample is hydrodynamically focused so that cells pass one by one through the laser beam.

Optics System: A series of lasers (commonly blue, red, and violet lasers) excite the fluorescent markers. Forward and side-scattered light, along with fluorescence emissions, are collected by detectors.

Detectors and Filters: Fluorescent signals emitted by the cells are separated by dichroic mirrors and filters into specific wavelengths for detection.

Electronics System: The emitted signals are converted into digital data, which is processed by a computer for analysis.

Data Collection:

Forward Scatter (FSC): Measures the size of the cell.

Side Scatter (SSC): Measures the internal complexity or granularity of the cell (e.g., nucleus, organelles).

Fluorescence Detection: Cells stained with fluorescent markers emit light when excited by lasers. The intensity of this emitted light correlates with the quantity of the target molecule bound by the fluorescent antibody.

Data Analysis:

The collected data is plotted and analyzed in a multi-dimensional format. The most common formats are:

Histograms: Display a single parameter (e.g., fluorescence intensity) on the x-axis and cell count on the y-axis. This shows the distribution of a single marker across the cell population.

Dot Plots: Display two parameters (e.g., FSC vs. SSC or fluorescence intensities of two markers) on the xand y-axes. Cells can be grouped into distinct populations based on the expression of multiple markers.

Gating: The process of selecting and isolating specific cell populations based on characteristics (e.g., size, complexity, or fluorescence). Gating helps focus on the subpopulations of interest.

 Key Steps in Flow Cytometry Analysis:

Cell Staining:

Cells are labeled with fluorochrome-conjugated antibodies specific to proteins of interest (e.g., CD markers for immune cells, tumor markers for cancer cells).

Multiple antibodies with different fluorescent labels can be used to analyze several parameters simultaneously (multicolor flow cytometry).

Compensation:

Since different fluorescent markers can overlap in emission spectra, compensation is applied to adjust for spectral overlap between different fluorophores. This ensures that the signals from each fluorophore are accurately measured.

Acquisition:

Cells pass individually through the laser beam, and the flow cytometer captures data for each cell based on its size, internal complexity, and fluorescence intensity.

Data for tens of thousands or millions of cells can be acquired in a matter of minutes.

Data Gating:

Gating refers to drawing regions on a dot plot or histogram to identify populations of interest, such as specific immune cell subsets or cancer cells.

Gating helps remove debris or dead cells and focuses analysis on live, viable cells.

Example: A common gating strategy for immune cells might start by gating lymphocytes based on forward and side scatter properties and then identify specific T-cell or B-cell populations based on CD markers.

Quantitative Analysis:

After gating, the percentage or absolute number of cells expressing certain markers is calculated.

Fluorescence intensity can provide information about the expression level of proteins (e.g., surface receptor expression levels).

Data Interpretation:

The analysis reveals information such as:

The proportion of cell subtypes (e.g., the percentage of CD4+ and CD8+ T cells in an immune sample).

The expression levels of proteins (e.g., upregulation or downregulation of specific markers).

Cell activation state, proliferation, or apoptosis (via specific markers like Annexin V for apoptosis).

 Applications of Flow Cytometry:

Immunophenotyping

Widely used to characterize immune cell populations by detecting specific surface or intracellular markers (e.g., CD4, CD8, CD19).

Important in diagnosing immune-related diseases (e.g., leukemia, lymphoma, HIV infection).

Cell Cycle Analysis:

Propidium iodide (PI) or DAPI can be used to stain DNA, allowing the determination of the cell cycle phase (G0/G1, S, G2/M).

Useful in cancer research to study cell proliferation.

Apoptosis Detection:

Flow cytometry can detect markers of apoptosis, such as Annexin V (for early apoptosis) and propidium iodide (for late apoptosis or necrosis).

Functional Studies:

Used to measure cellular functions such as cytokine production, phagocytosis, calcium flux, or oxidative burst.

Detect intracellular cytokines like IFN-γ, TNF-α, or IL-2 using permeabilization and intracellular staining techniques.

Cancer Research:

Flow cytometry is used for analyzing tumor markers, understanding cancer stem cells, and monitoring treatment response.

Helps in minimal residual disease (MRD) detection in leukemia and lymphoma.

Stem Cell Research:

Used for characterizing and isolating specific stem cell populations based on surface markers (e.g., CD34+ for hematopoietic stem cells).

Vaccine Development:

Flow cytometry can assess immune responses to vaccines by measuring the activation of specific immune cell subsets and cytokine production.

 Advantages of Flow Cytometry:

Multiparametric Analysis: Can simultaneously measure several characteristics (e.g., size, granularity, multiple markers).

High Throughput: Analyzes thousands to millions of cells quickly, making it suitable for large-scale studies.

Quantitative: Provides detailed quantitative data on protein expression and population frequencies.

Versatile: Can be adapted to various cell types and experimental conditions, making it a powerful tool in many research areas.

 Limitations:

Complexity: Data analysis can be complex, especially in multicolor experiments, requiring expertise in gating strategies and compensation.

Sample Preparation: Requires a single-cell suspension, which may not be easy to obtain from all tissue types.

Cost: Flow cytometers and reagents can be expensive, especially for high-parameter analyses.

Flow cytometry is an indispensable tool for studying cellular processes, immune profiling, and disease research, allowing for the detailed analysis of cell populations with high precision and throughput.

2310 月/24

KMD Bioscience-ELISA screening

Antibody Discovery

ELISA screening refers to using the Enzyme-Linked Immunosorbent Assay (ELISA) as a high-throughput method to screen a large number of samples for the presence of a specific antigen or antibody. This approach is commonly used in diagnostics, drug discovery, vaccine development, and research to identify the presence of proteins, antibodies, or other biomolecules in biological samples.

Types of ELISA Screening:

ELISA screening can be applied in different formats depending on the goal of the assay. Common types include:

  1. Antibody Screening: Identifying the presence of specific antibodies (e.g., screening for immune response in vaccine trials or detection of infectious diseases like HIV or COVID-19).
  2. Antigen Screening: Detecting specific antigens (e.g., pathogens, toxins, biomarkers) in biological samples.
  3. Compound Screening: In drug discovery, screening compounds for their ability to inhibit or enhance the interaction between an antigen and antibody.

 Steps for ELISA Screening:

Sample Collection and Preparation:

Collect biological samples, such as serum, plasma, urine, or cell culture supernatants.

Prepare the samples by diluting them in an appropriate buffer, ensuring that the antigen or antibody concentration falls within the dynamic range of the assay.

Coating the Microplate (Capture Antigen or Antibody):

For antibody screening, coat the microplate with the target antigen.

For antigen screening, coat the plate with an antibody that specifically binds the target antigen.

Incubate the plate for 1-2 hours or overnight, followed by blocking with a blocking buffer to prevent non-specific binding.

Addition of Samples:

Add prepared samples to the wells of the microplate.

In antigen screening, the target antigen in the sample will bind to the coated antibody.

In antibody screening, antibodies in the sample will bind to the immobilized antigen.

Incubate for 1-2 hours at room temperature or as optimized for your assay.

Addition of Detection Antibody:

After washing away unbound materials, add a detection antibody that specifically binds to the antigen (in antigen screening) or to the antibodies from the sample (in antibody screening).

The detection antibody can be directly conjugated to an enzyme (in direct ELISA), or a secondary antibody conjugated to an enzyme can be added in an indirect or sandwich ELISA.

Substrate Addition:

Add the enzyme substrate, such as TMB (3,3′,5,5′-tetramethylbenzidine) for HRP (horseradish peroxidase) or pNPP for alkaline phosphatase.

The enzymatic reaction generates a color change, which is proportional to the amount of bound antigen or antibody.

Signal Detection:

Measure the intensity of the color change using a microplate reader. The absorbance is measured at a specific wavelength (e.g., 450 nm for TMB).

Compare the absorbance values of the samples to a standard curve or a cutoff value to determine whether the target antigen or antibody is present in the sample.

 Applications of ELISA Screening:

Diagnostic Testing:

Infectious Disease Screening: ELISA is widely used for screening blood samples for pathogens like HIV, hepatitis viruses, and COVID-19.

Allergy Testing: ELISA screens for specific antibodies (IgE) in allergic patients.

Cancer Biomarker Screening: ELISA can detect biomarkers like PSA (prostate-specific antigen) for cancer diagnosis.

Vaccine Development and Monitoring:

ELISA screening is used to evaluate the immune response in vaccinated individuals by detecting specific antibodies against the vaccine antigen.

Researchers use ELISA to screen sera from vaccine trial participants to assess vaccine efficacy.

Drug Discovery and Pharmacokinetics:

In drug discovery, ELISA is used to screen potential drug candidates for their effect on antigen-antibody interactions.

ELISA is also employed to measure drug levels, such as therapeutic antibodies, in biological samples during pharmacokinetic studies.

Environmental and Food Safety:

ELISA can screen for contaminants such as toxins, pesticides, and allergens in food and environmental samples.

Research:

ELISA screening is commonly used in research to screen for cytokines, growth factors, and other proteins involved in cellular processes.

 Advantages of ELISA Screening:

High-Throughput: ELISA allows for the screening of hundreds of samples in a single 96or 384-well plate, making it ideal for large-scale studies.

Quantitative or Qualitative: ELISA can provide quantitative data on the amount of antigen or antibody present or qualitative results for positive/negative screening.

Sensitive and Specific: ELISA is highly sensitive and specific, particularly when well-optimized antibodies and conditions are used.

 Key Considerations for ELISA Screening:

Cutoff Values: In screening applications, it is crucial to define a reliable cutoff value to distinguish between positive and negative samples.

Controls: Include appropriate positive and negative controls to ensure the accuracy of the screening results.

Sample Dilution: Ensure that the antigen or antibody concentration in the samples is within the linear range of the assay. This may require optimizing dilution factors.

Reproducibility: Run samples in duplicates or triplicates to ensure reproducibility and reliability of the screening results.

Validation of ELISA Screening:

Sensitivity and Specificity: Validate the assay to ensure it can reliably detect low levels of the target molecule without generating false positives or negatives.

Precision: Evaluate intraand inter-assay variability to ensure consistent results across different runs.

Linearity: Test a range of concentrations to confirm that the assay produces linear results within the target concentration range.

ELISA screening is a versatile and reliable method that plays a key role in diagnostics, research, and drug development. With proper optimization, it can provide highly accurate results for detecting antigens, antibodies, or other molecules across large numbers of samples.

2210 月/24

KMD Bioscience-Elisa Assay Development

CRO Service

Enzyme-Linked Immunosorbent Assay (ELISA) development involves several critical steps to create a sensitive, specific, and reproducible assay for detecting proteins, peptides, antibodies, or hormones in biological samples. The process of developing a successful ELISA requires optimizing each step for the target analyte. Here’s a detailed guide to the steps involved in developing an ELISA assay:

 Types of ELISA Assays:

Direct ELISA: Involves detecting an antigen immobilized on a plate using an enzyme-conjugated primary antibody.

Indirect ELISA: Uses a primary antibody to detect the antigen, followed by an enzyme-conjugated secondary antibody for detection.

Sandwich ELISA: The target antigen is “sandwiched” between a capture antibody and a detection antibody.

Competitive ELISA: Measures antigen concentration by competing with labeled antigen for a limited amount of antibody binding sites.

Steps for ELISA Assay Development:

Selection of Target and Antibodies

Target Antigen: Define the target of interest (e.g., protein, peptide, hormone, etc.).

Antibody Selection:

Monoclonal antibodies provide high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes.

For a sandwich ELISA, use two antibodies (capture and detection) that bind to different epitopes on the same antigen.

Verify the antibodies for specificity, sensitivity, and cross-reactivity.

Coating the Microplate (Capture Antibody or Antigen)

Coat a 96-well microplate with either a capture antibody (for sandwich ELISA) or the antigen (for direct or competitive ELISA).

Use coating buffer (e.g., carbonate-bicarbonate buffer, pH 9.6) to bind antibodies or antigens to the wells.

Incubation: Coat the plate overnight at 4°C or for 1-2 hours at 37°C.

After coating, block any remaining binding sites to prevent non-specific binding.

Blocking

Blocking minimizes non-specific binding and reduces background noise by covering unbound sites on the microplate.

Common blocking agents include:

Bovine serum albumin (BSA)

Non-fat dry milk

Casein

Tween-20 detergent

The blocking step usually takes 1-2 hours at room temperature or can be done overnight at 4°C.

Sample and Standard Preparation

Prepare samples (e.g., serum, plasma, cell lysates) and standards (known concentrations of the target antigen).

Dilute samples appropriately using the sample diluent to ensure the antigen concentration falls within the dynamic range of the assay.

Adding the Samples and Standards

Add samples and standards to the wells and incubate for 1-2 hours at 37°C (or as required).

During incubation, the target antigen will bind to the coated capture antibody or antigen.

Addition of Detection Antibody

After washing away unbound material, add the detection antibody (either enzyme-conjugated or unlabelled).

If the detection antibody is unlabeled (for indirect or sandwich ELISA), you will add an enzyme-conjugated secondary antibody after incubation.

Incubation: Allow time for the detection antibody to bind to the target (30 minutes to 2 hours, depending on the assay).

Washing

After each incubation step (sample addition, antibody addition), wash the plate multiple times with wash buffer (commonly PBS or Tris-buffered saline with 0.05% Tween-20).

Adequate washing reduces background signal and prevents non-specific binding.

Enzyme-Conjugated Secondary Antibody (if applicable)

For indirect or sandwich ELISA, add the enzyme-linked secondary antibody after washing.

The secondary antibody binds to the detection antibody, amplifying the signal.

Substrate Addition

Add the substrate specific to the enzyme conjugated to the detection antibody. Common enzyme-substrate systems include:

Horseradish peroxidase (HRP) with TMB (3,3′,5,5′-tetramethylbenzidine) substrate (produces a blue color).

Alkaline phosphatase (AP) with p-nitrophenyl phosphate (pNPP) substrate (produces a yellow color).

Allow time for the color development (usually 10-30 minutes).

Stop the reaction using a stop solution (e.g., sulfuric acid for TMB).

Detection and Data Analysis

Measure the intensity of the color change using a microplate reader at the appropriate wavelength (e.g., 450 nm for TMB-HRP).

Generate a standard curve using the known concentrations of antigen from the standards.

Use the standard curve to quantify the antigen concentration in the samples.

Optimization Steps for ELISA Development:

Antibody Concentration: Optimize the concentration of both capture and detection antibodies to achieve the best signal-to-noise ratio.

Blocking and Washing Conditions: Ensure blocking conditions prevent non-specific binding, and optimize wash steps to minimize background noise.

Incubation Times: Optimize the duration of incubation steps for both antigen/antibody binding and enzyme-substrate reactions.

Substrate and Enzyme Reactions: Select a substrate with high sensitivity, and optimize the time for color development for maximum signal without over-developing.

 Validation and Troubleshooting:

Sensitivity and Specificity: Test the assay with known positive and negative controls to ensure sensitivity and specificity.

Dynamic Range: Verify the linearity of the assay by testing a range of concentrations to ensure the assay can detect a wide range of antigen concentrations.

Reproducibility: Perform repeat assays to assess intraand inter-assay variability.

Applications of ELISA:

Diagnostics: Used in detecting disease markers, hormones, or pathogens in clinical samples.

Drug Development: ELISA is employed for pharmacokinetics, therapeutic antibody detection, and biomarker studies.

Research: Widely used for detecting cytokines, growth factors, and other proteins in biological samples.

A well-developed ELISA assay can provide a reliable, sensitive, and specific tool for quantifying target molecules in research, diagnostics, and drug development.

1710 月/24

KMD Bioscience-Yeast One-hybrid Assay

Protein Engineering

The Yeast One-Hybrid (Y1H) Assay is a molecular biology technique used to identify protein-DNA interactions. While the Yeast Two-Hybrid (Y2H) assay focuses on protein-protein interactions, the Y1H assay is specifically designed to determine whether a particular protein binds to a specific DNA sequence. This makes it a valuable tool for identifying transcription factors that regulate gene expression by binding to promoter or enhancer regions in DNA.

How the Yeast One-Hybrid Assay Works:

DNA Sequence as Bait:

A specific DNA sequence of interest, such as a promoter or regulatory element, is inserted into a reporter plasmid. This DNA sequence is used as the “bait.” It is placed upstream of a reporter gene (e.g., lacZ or HIS3), which allows for the detection of binding interactions.

The bait plasmid is then integrated into the yeast genome.

Protein as Prey:

The prey proteins (usually a library of potential transcription factors or other DNA-binding proteins) are fused to an activation domain (AD). These fusion proteins are introduced into yeast cells.

If a prey protein binds to the bait DNA sequence, the AD will activate transcription of the reporter gene.

Reporter Gene Expression:

If the prey protein binds to the bait DNA sequence, it will bring the AD close enough to the transcription machinery to activate the reporter gene.

Reporter gene activation allows for easy detection through:

Growth on selective media (e.g., HIS3 reporter allows growth on histidine-deficient media).

Colorimetric assays (e.g., lacZ reporter leads to blue coloration in the presence of X-gal).

Identification of DNA-Binding Proteins:

Positive yeast colonies that show reporter gene expression (growth or color change) are selected.

The DNA of the prey plasmid is then sequenced to identify the protein that is responsible for binding the DNA bait sequence.

Applications of the Yeast One-Hybrid Assay:

Identification of Transcription Factors:

Y1H is commonly used to identify transcription factors that bind to specific promoter or enhancer sequences. This helps researchers understand how genes are regulated at the transcriptional level.

Characterization of Regulatory Elements:

By using different regulatory DNA sequences as bait, researchers can identify proteins that interact with those sequences and contribute to gene regulation.

Studying Protein-DNA Interactions in Different Organisms:

Although the assay is performed in yeast, it can be used to study protein-DNA interactions from different organisms, including humans, plants, and bacteria, by expressing their transcription factors in yeast.

Regulatory Network Mapping:

The Y1H assay can be used to map regulatory networks, where multiple transcription factors bind to different regulatory elements of various genes. This helps in understanding how complex gene expression is controlled.

Advantages of the Yeast One-Hybrid Assay:

Efficient and Scalable: Y1H allows for high-throughput screening, making it an efficient way to identify many DNA-binding proteins from large libraries.

In Vivo Environment: Protein-DNA interactions are detected in a eukaryotic (yeast) cellular environment, which often makes it more biologically relevant than in vitro techniques.

Specificity: Since the assay directly detects binding to specific DNA sequences, it provides specific insights into transcription factor-DNA interactions.

Limitations of the Yeast One-Hybrid Assay:

False Positives: Some proteins may bind non-specifically to DNA sequences, leading to false positive results.

False Negatives: The assay may not detect interactions if the transcription factor requires post-translational modifications or if it is not properly folded in yeast cells.

Yeast-Specific Context: Some proteins or transcription factors from other organisms may not function properly in yeast, potentially missing important interactions.

 Example:

To study a transcription factor that regulates the expression of a gene related to stress response, researchers might:

  1. Use the promoter region of that gene as the bait in the Y1H assay.
  2. Screen a library of transcription factors (prey) to identify which ones bind to that promoter.
  3. The identified transcription factors could be further studied to understand their role in the gene’s regulation.

 

The Y1H assay is a powerful method for studying gene regulation by identifying transcription factors and other DNA-binding proteins, contributing significantly to our understanding of cellular regulation and development.

1610 月/24
Recombinant protein production

KMD Bioscience-Protein Assay Protocol

Protein Engineering

Protein assays are used to measure the concentration of proteins in a sample, which is essential for downstream applications such as enzyme assays, Western blotting, and protein purification. Several protein assays exist, with the Bradford assay, BCA assay, and Lowry assay being the most commonly used. Below is a detailed protocol for the Bradford protein assay, a widely used and rapid method based on the binding of Coomassie Brilliant Blue dye to proteins.

Bradford Protein Assay Protocol

Materials Needed

Bradford reagent: Commercially available as a ready-to-use solution or prepared by dissolving Coomassie Brilliant Blue G-250 in phosphoric acid and ethanol.

Protein standard: Bovine Serum Albumin (BSA) or other known protein for generating a standard curve.

Unknown protein samples.

Distilled water or buffer: Used to dilute the protein samples and standards.

Microplate reader or spectrophotometer: Set to measure absorbance at 595 nm.

Cuvettes or 96-well microplate: For sample measurement.

Pipettes and tips.

Preparation of Bradford Reagent

If not using a commercially available Bradford reagent, it can be prepared by:

Dissolving 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of ethanol.

Adding 100 mL of 85% phosphoric acid.

Diluting to 1 liter with distilled water.

Filter the solution and store in an amber bottle at room temperature.

Step-by-Step Procedure

  1. Prepare Protein Standards

Prepare a series of BSA standards (or another standard protein) by serially diluting a known concentration of BSA (e.g., 1 mg/mL) to generate a range of concentrations, typically between 0–1 mg/mL.

Example standard curve dilutions:

0 µg/µL (blank)

2 µg/µL

4 µg/µL

6 µg/µL

8 µg/µL

10 µg/µL

Dilute the BSA standards with distilled water or the same buffer used in the unknown protein samples.

  1. Dilute the Unknown Protein Samples

If the concentration of the unknown protein is unknown, make serial dilutions of your sample to ensure that the protein concentration falls within the range of the standard curve.

For high-concentration protein samples, you may need to dilute the sample in water or buffer.

  1. Add Bradford Reagent

Microplate method:

Pipette 10 µL of each protein standard and unknown sample into separate wells of a 96-well plate.

Add 200 µL of Bradford reagent to each well.

Mix the contents of each well by gently tapping the plate or using a plate shaker.

Cuvette method:

Pipette 20 µL of each protein standard and unknown sample into cuvettes.

Add 1 mL of Bradford reagent to each cuvette and mix thoroughly.

  1. Incubate the Reaction

Allow the reaction to incubate at room temperature for 5–10 minutes, but no longer than 1 hour.

During this time, the Coomassie dye binds to the protein, shifting its absorption maximum from 465 nm to 595 nm, resulting in a color change from brownish-red to blue.

  1. Measure Absorbance

Use a microplate reader or spectrophotometer set to 595 nm to measure the absorbance of each well or cuvette.

Measure the absorbance of the blank (0 µg/µL BSA) and subtract this value from all other absorbance values to account for background absorbance.

  1. Generate a Standard Curve

Plot the absorbance values of the BSA standards against their known concentrations to generate a standard curve.

The plot should show a linear relationship between protein concentration and absorbance within the range of the assay.

Fit a trendline to the standard curve and use the equation of the line (typically y = mx + b, where y is absorbance, x is protein concentration, m is the slope, and b is the intercept) to determine the concentration of protein in the unknown samples.

  1. Calculate Protein Concentration of Unknown Samples

Use the absorbance values of the unknown samples to interpolate their concentrations from the standard curve.

Apply the equation obtained from the standard curve to calculate the protein concentration of each unknown sample:

Protein concentration (µg/µL) = (Absorbance b) / m

Multiply by the dilution factor if the unknown sample was diluted before performing the assay.

 Notes:

Linear range: The linear range of the Bradford assay is typically between 0.1 to 1.0 mg/mL. If the absorbance of an unknown sample is above this range, dilute the sample and repeat the measurement.

Interfering substances: Detergents (e.g., SDS), high concentrations of salts, and certain buffers (e.g., Tris) can interfere with the Bradford assay. If interference is suspected, switch to an alternative assay like the BCA assay.

Optional: BCA Assay Protocol

For proteins in detergents or other problematic solutions, the BCA (Bicinchoninic Acid) Assay may be more suitable. This assay is compatible with detergents and measures protein concentration by producing a purple-colored complex with proteins that absorb at 562 nm.

Troubleshooting

Low absorbance values: If absorbance values are too low, check that the protein concentration is within the standard curve range. If not, concentrate the sample or prepare fresh standards.

High absorbance values: If absorbance values are too high, dilute the protein samples to bring them within the assay’s linear range.

By following these steps, you can accurately determine the concentration of protein in your samples using the Bradford protein assay.