Common Troubleshooting Tips for Western Blot Results (part 1)

A: They have different functions: Positive control lysate demonstrates that the staining protocol is successfully performed and gives the expected level of sensitivity/specificity as characterized during technical optimization, while negative control lysate checks for non-specific binding. (false-positive results)

Commonly used positive controls include:

Samples from cells exhibiting overexpression of target protein

Cell line/tissue/experimental condition with a proven positive signal

Purified recombinant protein

Commonly used negative controls include:

Samples from knockdown or knockout tissue/cell lines

Samples from RNA interference targeted lines

Table. Established controls for Western Blot

A: Compared to theoretical molecular weight, molecular weight has three dimensions to change: increase, decrease, or change to a specific number.

Molecular Weight increase may be caused by these factors:

1: Post-translational modifications (PTMs): Modifications like phosphorylation or glycosylation can increase a protein's molecular size.

Solution: Analyze the protein using deglycosylation enzymes or phosphatase to confirm PTMs.

2: Protein Refolding and Multimerization: Denatured proteins may refold and form dimers, trimers, or tetramers, resulting in molecular weights that are two, three, or four times the monomer size.

Solution: Use reducing and denaturing conditions during SDS-PAGE to break down multimers.

Molecular Weight decrease may be caused by these factors:

1: Protein Cleavage After Activation: Many proteins are synthesized as precursors and cleaved into active fragments, leading to a smaller actual molecular weight.

Solution: Use specific antibodies targeting precursor and active forms to verify cleavage.

2:Protein Degradation: Partial degradation of proteins can produce fragments with lower molecular weights.

Solution: Use protease inhibitors during sample preparation to prevent degradation.

In some cases, the molecular weight may change to a specific number:

1: Differences in Apparent and Theoretical Molecular Weight: Some proteins, such as p53, have an apparent molecular weight (e.g., 53 kDa, from which its name derives) that differs from the theoretical value calculated from its amino acid sequence (e.g., 43 kDa for p53).

Solution: Verify molecular weight discrepancies by cross-referencing with literature or databases.

2: Multiple Isoforms: Many proteins exist in various isoforms, each with a different molecular weight.

Q3: What causes the high background in my western blot results and how can I solve it?

A: Here are some common possible causes and suggestions.

1: Inadequate Membrane Blocking

Solution: Extend the blocking time or choose a more suitable blocking buffer.

2: Inappropriate Primary Antibody Dilution

Solution: Perform gradient testing to find the optimal antibody dilution and reduce antibody concentration.

3: High Primary Antibody Incubation Temperature

Solution: Incubate at 4°C overnight.

4: Overexposure During Detection

Solution: Reduce the exposure time.

5: Insufficient Membrane Washing

Solution: Increase the washing time or wash the membrane more frequently.

Q4: What causes the multiple non-specific bands in western blot results and how can I solve it?

A: Here are some common possible causes and suggestions.

1: Target Protein Has Multiple Modification Sites (e.g., Phosphorylation, Glycosylation, Acetylation):

Solution: Review the literature or conduct bioinformatics analysis to identify modification sites. Remove modifications to confirm the protein's true size.

2: Target Protein Has Alternative Splicing Variants:

Solution: Consult literature or perform bioinformatics analysis to determine the possibility of splicing variants.

3: Degradation of Target Protein During Sample Preparation:

Solution: Use protease inhibitors and handle samples on ice.

4: Excessive Loading Amount or High Sensitivity:

Solution: Reduce the sample loading amount appropriately.

5: High Concentration of Primary or Secondary Antibody:

Solution: Lower the antibody concentration.

Next up

In part 2, we will address the following questions:

Lack of signal or weak signal in Western blot results.

Inconsistent protein band intensity in WB experiments.

Skewed or drifting bands in WB results.

For each, we will explore the potential causes and provide solutions.