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.