Collect and prepare your protein samples of interest. This may involve cell lysis, tissue homogenization, or protein extraction.
Determine the protein concentration using a suitable assay (e.g., Bradford assay, BCA assay) and adjust the concentrations of your samples accordingly.
2. Gel Electrophoresis
Prepare a polyacrylamide gel with an appropriate percentage depending on the size of the proteins you want to separate (typically 8-12% for most applications).
Load the protein samples onto the gel, along with a molecular weight marker for reference.
Run the gel using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) under constant voltage until the proteins have migrated to their desired positions.
3. Protein Transfer
Prepare a transfer apparatus with a suitable membrane (typically nitrocellulose or PVDF) that matches the size of your gel.
Cut the gel carefully to separate the stacking and resolving gel portions.
Activate the membrane by soaking it in methanol for a few seconds and then transfer it to transfer buffer (e.g., Towbin buffer or a semi-dry transfer buffer).
Assemble the transfer sandwich: place the gel and membrane on the anode side, followed by filter papers, and then the cathode.
Perform the protein transfer using a suitable transfer method (wet, semi-dry, or tank transfer) according to the manufacturer's instructions.
Incubate the membrane in blocking solution (e.g., 5% non-fat dry milk or 3% bovine serum albumin) for about 1 hour at room temperature or overnight at 4°C to block non-specific binding sites.
5. Primary Antibody Incubation
Dilute your primary antibody of interest in a suitable antibody dilution buffer (often TBS with 0.1% Tween-20 or TBS-T) according to the manufacturer's recommendations.
Incubate the membrane with the primary antibody solution for 1-2 hours at room temperature or overnight at 4°C on a gentle rocking platform.
6. Washing
Wash the membrane multiple times with TBS-T to remove unbound primary antibody. Typically, 3-5 washes of 5-10 minutes each are performed.
7. Secondary Antibody Incubation
Dilute an appropriate secondary antibody (conjugated to an enzyme such as horseradish peroxidase) in antibody dilution buffer.
Incubate the membrane with the secondary antibody solution for 1-2 hours at room temperature on a gentle rocking platform.
8. Washing
Wash the membrane again with TBS-T as in step 6 to remove unbound secondary antibody.
9. Visualization
Prepare a suitable substrate solution for the enzyme-conjugated secondary antibody (e.g., chemiluminescent or chromogenic substrate).
Apply the substrate solution to the membrane and incubate for the recommended time.
Image the blot using an appropriate detection system (e.g., chemiluminescence imager or X-ray film) to visualize the protein bands.
10. Quantification and Analysis
Analyze the band intensities using image analysis software or other quantification methods.
Compare the protein bands of interest between different samples, and perform statistical analysis if required.
Remember to always refer to the specific protocols and instructions provided by the manufacturers of the reagents.
