Western Blot Protocol | Complete Guide with Tips on Troubleshooting

Western blotting is a sensitive immunoassay reaction technique that can detect protein molecules at nano gram quantities.

It is a widely used technique in cellular and molecular biology for the detection and identification of a specific proteins from the complex mixtures.

This is based on the application of an electrical current which supports the protein to move from gel to the membrane.

Western Blot Protocol - blotting technique

 

Principle

The principle of western blot is based on the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to sheets of polyvinylidene difluoride (PVDF) or nitrocellulose membrane, followed by immuno detection of proteins using antibodies with fluorescent or chemiluminescent detection.

Technique

1 SDS phase unit: Separation of proteins is done based on their size. First heavyweight proteins, then the smaller ones and finally the smallest ones at last.
2 Transfer unit: Proteins from SDS gels to PVDF or nitrocellulose membrane- under controlled temperate (40C).
3 Identification of specific protein by primary and secondary antibody: densitometric analysis of protein expression.

Chemicals and Solution

Lysis buffer Loading buffer Running buffer 10X Transfer buffer
RIPA buffer
150 mM NaCl 20% glycerol 25 mM Tris base 25 mM Tris base
50 mM Tris-HCl, pH 8.0 10% 2-mercaptoethanol 190 mM glycine 190 mM glycine
0.1% Triton X-100 4% SDS 0.1% SDS 20% methanol
0.5% sodium deoxycholate 0.004% bromophenol blue the pH adjust to 8.3 Check the pH and adjust to 8.3
0.1% SDS 0.125 M Tris-HCl
Protease inhibitors the pH adjust to 6.8

Blocking buffer
3–5% milk or BSA (bovine serum albumin)
Add to TBST buffer.

Protocol
Tissues lysate preparation
1. Tissue dissection should be on ice pack with controlled temperature to avoid deterioration from proteases.
2. The tissue must be placed on Eppendorf tubes or another suitable tube, minse the tissue mass and homogenize then add ~300 μL of ice cold lysis buffer according to the amount of tissue present. Protein should not be too diluted, to minimize the risk of exclusion.
3. The minimum to optimal concentration range is 0.1-5 mg/mL.
4. Centrifuge at 12,000 rpm, 4°C for 10 min in a micro-centrifuge. Remove the tube and the supernatant transfer to a fresh tube; the process should be on an ice pack, discard the pellet.

Sample preparation
1. Protein concentration determination for loading of an equal volume of protein using 2X Laemmli sample buffer.
2. Denaturation of protein by putting sample buffer at 100°C for 5 min. The sample must be divided into aliquots and stored at -20°C for future use.

Loading and running the gel
1. Load an equal quantity of protein into an SDS-PAGE gel, with the latter as a molecular weight marker.
2. Load 20–30 μg of total protein from tissue homogenate.
3. The gel runs at 100 V for 1-2 hrs.

Transfer of protein to the membrane
After the electrophoretic transfer of protein, transfer to membrane require
1 PVDF or nitrocellulose membrane can be used to transfer of protein from gel to a membrane
2 Initially, expose the membrane with methanol for one min and then rinsed with transfer buffer
3 After activation make the stacks using blotting paper
4 Fix the transfer unit and put the set up at 40C
5 Run transfer unit at 50 volts for 3 hrs.

Antibody staining
1. After the transfer steps remove the membrane and wash with test buffer
2. Block the membrane for 1hr with blocking buffer.
3. Incubate for overnight with primary antibody (1:1000) at 4o c on gentle shaking.
4. Wash with 1X TBST 3 times with vigorous shaking.
5. Incubate for 1hr with secondary antibody (1:1000) at room temperature on gentle shaking.
6. Wash with 1X TBST 3 times with vigorous shaking.
7. Use approx 50-100μl of ECL( BIORAD 1:1 reagent mixed) reagent on the membrane.
8. Develop the blots using a Chemi-DOC.
9. Normalize the bands with housekeeping genes and quantify the bands using IMAGE-J.

Troubleshooting Guide

Problemcausesolution
Non specific peak or uneven bandno color change observed during enzyme substrate conjugationCheck conjugate and substrate concentration and verify it. It should change color
Incorrect substrate for applicationConfirm that the substrate selected is appropriate for conjugation with the specific enzyme.
primary and secondary antibody dilutionfollow the instruction change in dilution will affect the band or peak intensity
Inappropriate primary and secondary antibodyassured the species in which primary antibody raised and select other species raised secondary antibody
Protein detection not occurredcheck the protein concentration to be loaded and increased it if required
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