Western Blot Troubleshooting Guide: Key Solutions & Tips Guide

WB experiment (also known as Western Blot experiment or Western blot), the essence is the specific reaction of antigen and antibody, the basic principle is to separate denatured proteins through SDS polyacrylamide gel electrophoresis and transfer it to a solid phase carrier (such as PVDF membrane, NC membrane) via membrane transfer (wet or semi-dry rotation). Following blocking (using BSA, buttermilk, etc.), the primary antibody is applied to bind specifically to the protein, and the fluorescein-labeled secondary antibody is added after washing the film. by binding the second antibody with the primary antibody, the target protein can be observed by substrate color development and chemiluminescence. It is generally used to determine whether a specific protein is expressed in the sample and roughly analyze its expression level.

The WB experiment is often used for verifying protein expression to detect protein-protein interactions, protein-DNA interactions, post-translational modifications, etc., which are significant for protein separation and purification. The solution label incorporated into the expression vector can be confirmed through Western blot assay to streamline subsequent protein separation and purification processes.

As the design process of the Western Blot experiment is cumbersome and time-consuming, various issues may arise from sample preparation to development, necessitating attention to numerous experimental details. Common problems in Western blot experiments typically include the following aspects:

Q 1: The Destination Strip Is Not Being Detected Or The Signal Is Weak.

Reason Analysis:

1. Protein Degradation.

A 1: Add a protease inhibitor or phosphatase inhibitor when preparing the sample, and store the sample separately to avoid repeated freeze-thaw cycles.

2. Protein intake is too low.

A 2: A higher concentration of loading buffer can be selected to avoid protein concentration reduction while increasing the loading volume. A positive control group was established to eliminate operational problems and enhance the accuracy of protein expression verification.

3. Small proteins are lost due to long electrophoresis time.

A 3: Reduce the electrophoresis time or voltage.

4. Electrophoresis or film transfer instrument power supply is reversed.

A 4: Make sure the electrodes are properly connected through inspection.

5. PVDF membrane is not activated.

A 5: Activate the PVDF membrane with methanol/ethanol for 1min then set it aside.

6. Insufficient transmembrane.

A 6: Reformulation of transmembrane fluid; Confirm the appropriate transfer time.

7. Primary or secondary antibody.

A 7: Check whether the species of the primary and secondary antibodies are the same, or replace the primary antibody to avoid failure.

8. Washing film for a more or longer time.

A 8: Appropriately reduce the number and time of film washing.

Q 2: The Strip Is Fuzzy And Trailing.

Reason Analysis:

1. Gel plate.

A 1: Gel formulated with unclean or undried gel plates is prone to bubbles.

2. Sample delivery.

A 2: The protein sample is fully dissolved to prevent absorption, precipitation, and cross-contamination.

3. Transmembrane.

A 3: The bubble is not removed or the "sandwich" is not tightened when the film is transferred resulting in the film is not sufficient.

Problem 3: The Background Is Too High.

Reason Analysis:

1. Inadequate closure.

A 1: Extend the sealing time or increase the concentration of the sealing liquid.

2. Washing is not sufficient.

A 2: Increase the time and frequency of washing to remove non-specific binding.

3. The exposure time is too long.

A 3: Reduce exposure time.

Problem 4: Multiple strips appear or the positions of the strips are incorrect.

Reason Analysis:

1. Inadequate closure.

A 1: Extend the sealing time or increase the concentration of the sealing liquid.

2. The specificity of the primary antibody is poor.

A 2: Replace the primary antibody.

3. The concentration of the secondary antibody is higher.

A 3: Increase the dilution ratio and reduce the antibody dosage of the secondary antibodies.

4. There are splinters in the target protein.

A 4: In the presence of splinters, the digestion of cells was reduced before protein extraction, and sequences and sites were queried by BLAST.

Problem 5: The Strips Are Uneven.

Reason Analysis:

1. The problem of making glue.

A 1: Select and make the appropriate concentration of glue, and pull out the comb smoothly after the glue sets, which prevents the strips from arching due to the convex point sample hole surface.

2. The amount of protein sample.

A 2: Reduce the amount of protein in the sample to avoid the sample appearing in other holes.

3. The electrophoresis speed is too fast.

A 3: Reduce the electrophoresis speed to ensure that proteins can be adequately isolated and localized to avoid the "smile" strip.

4. Electrophoresis temperature is too high.

A 4: Adjust the temperature of the electrophoresis system to avoid the phenomenon of the fuzzy strip, because the temperature is too high that can’t accurately separate proteins of different sizes.

KMD Bioscience has been committed to protein research for many years, providing protein interaction services for Western Blot experiments, Pull-down experiments, etc., and protein separation and purification services. KMD Bioscience has extensive experience in protein interaction experiments of design, which can save customers time and meet their needs.

References:

[1] Pillai-Kastoori L, Heaton S, Shiflett SD, et al. Antibody validation for Western blot: By the user, for the user [J]. J Biol Chem. 2020, 295(4): 926-939.

[2] Ferguson RE, Carroll HP, Harris A, et al. Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies [J]. Proteomics. 2005, 5(2): 566-571.

[3] Ren G, Juhl M, Peng Q, et al. Selection and validation of reference genes for qPCR analysis of differentiation and maturation of THP-1 cells into M1 macrophage-like cells [J]. Immunol Cell Biol. 2022, 100(10): 822-829.