37 Western Blotting Troubleshooting Tips

Western blotting is a technique used to determine the presence or absence of selected proteins in a sample. This online western blotting troubleshooting guide provides solutions for problems such as no bands observed, poor quality transfer and high background. Our guide equips researchers with comprehensive solutions and suggestions to help solve western blotting challenges.

No Bands Observed

Problem Explanation

1. Incorrect primary antibody

Antibody has low to no affinity

2. Inactive antibody

Perform a dot blot

3. Insufficient protein concentration

Increase the amount of protein and use a positive control

4. Poor transfer

Make sure the membrane is activated. Transfer buffer must contain methanol when using nitrocellulose membranes. PVDF membranes must be pre-soaked with methanol

5. Suboptimal transfer time

High molecular weight proteins may require longer transfer times

6. Incorrect secondary antibody

Confirm host species and IgG type of primary

7. Antibodies expired

Check that all antibodies are in date

8. Incorrect storage of antibodies

Ensure all antibodies are stored as per manufacturer’s instructions

9. Sodium Azide contamination

Sodium Azide contamination will quench HRP signal

10. Suboptimal primary antibody incubation time

Increase incubation time with the primary antibody

11. Incompatible primary and secondary antibody

Maintain a consistent species in both antibodies

12. Insufficient secondary antibody concentration

Increase the concentration of primary/secondary antibody

13. Excessive washing

Reduce the number and duration of washes

14. Incorrect orientation

Mark your membrane to ensure correct orientation

Poor Quality Transfer

Problem Explanation

15.   Membrane choice

Choose either PVDF/nitrocellulose membranes according to the target protein molecular weight

16.   Dry membrane

It is important not to let the membrane or filter paper dry out

17.   Incomplete protein resolution

Ensure optimal gel concentration is used for the protein of interest

18. Incorrect sample preparation

The sample must contain DTT or B-Mercaptoethanol and be heated prior to loading

High background

Problem Explanation

19.   Unspecific antibody binding

Ensure the correct and most specific primary antibody is used

20.   Insufficient blocking

Optimise blocking time duration

21.   Suboptimal antibody concentration

Optimise antibody concentration

22.   Insufficient washing

Increase the number of washes performed. Increase the concentration of Tween 20 used in wash buffer

23.   Incorrect membrane choice

Nitrocellulose membranes generally have less background compared to PVDF

24.   Film overexposed

Reduce the exposure time

Too Many Bands

Problem Explanation

25.   Unspecific antibody

Ensure the antibody used is specific for the protein of interest

26.   Proteolytic breakdown

Use protease inhibitors to prevent the proteolytic breakdown of the antigen

27.   Gel overloading

Overloading the gel with too much protein can cause the development of “Ghost bands.” Optimise protein concentration.

28.   Insufficient blocking

Extend the blocking time

29.   Low antigen concentration

Consider immunoprecipitating target protein

30.   Unspecific secondary antibody binding

Use secondary antibody only control. If bands develop use a different secondary antibody

31.   Analyte aggregation

Increase DTT concentration

32. Post translational modification

Protein sample has multiple modified forms e.g. acetylation, methylation and phosphorylation

33.   Protein degradation

Target protein of interest degraded

34.   Splice variants

Could lead to the visualisation of multiple bands

35.   High primary antibody concentration

Use a lower concentration of primary antibody


Problem Explanation

36. Unresolved proteins

Inefficient separation. Use high molecular weight and low molecular weight proteins

37.   Smile/Curve effect on the gel

Incorrect voltage. Inconsistent temperatures

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