Today, let's talk about the problem that has caused countless research colleagues headaches - Western Blot (WB) experiment. This seemingly simple but full of "traps" experiment, why does it always make us stumble again and again? Now let's review the reasons for failures together.
I. No bands
Possible reasons may include:
- Antibody issues: The antibody may be ineffective, nonspecific, or not binding to the target protein.
- Sample issues: The sample may contain interfering substances, be too low in concentration, or have already degraded.
- Electrophoresis issues: Inappropriate electrophoresis conditions, such as voltage, time, or improper use of buffer.
- Transfer issues: Problems during the transfer process, such as inappropriate membrane selection, transfer time, or transfer buffer.
- Blocking issues: The blocking solution may be inappropriate or the blocking time may be too long.
- Staining issues: The staining reagent may be expired, insensitive, or improperly used.
Equipment issues: The equipment may malfunction or not be properly calibrated.
- Experimental operation issues: The experimental operation may not be standardized, such as insufficient washing or uneven sample loading.
- Protein modification issues: The target protein may undergo post-translational modifications that prevent antibody recognition.
- Target protein expression issues: The target protein may have very low expression or no expression in the cells or tissues being detected.
II. High background
Possible reasons may include:
- High antibody concentration: Using too much antibody may increase nonspecific binding, resulting in high background.
- Antibody quality issues: The antibody may have high nonspecific binding or cross-react with other proteins.
- Insufficient blocking: The blocking solution fails to effectively cover nonspecific binding sites on the membrane, resulting in high background.
- Membrane issues: The membrane may have poor quality or contamination, leading to high background.
- Incomplete transfer: During the transfer process, proteins may not be fully transferred onto the membrane, resulting in high background.
- Insufficient washing: The washing steps fail to effectively remove unbound antibody or other impurities, leading to high background.
- Staining reagent issues: The staining reagent may be too sensitive or unstable, resulting in high background.
- Experimental equipment issues: The equipment may have leakage or interference, leading to high background.
- Protein sample issues: The sample may contain a large amount of interfering substances, such as protein degradation products or other impurities.
- Experimental operation issues: The experimental operation may not be standardized, such as prolonged incubation time or high temperature.
III. Nonspecific bands or incorrect protein band sizes
Possible reasons may include:
- Too many passages of cells, resulting in different protein expression levels, leading to inconsistent band sizes or the presence of multiple bands.
- Protein sample degradation, resulting in lower protein molecular weight or the presence of multiple bands.
- In vivo expressed protein samples may have various forms of modifications such as acetylation, methylation, alkylation, phosphorylation, and glycosylation, leading to inconsistent band sizes or the presence of multiple bands.
- The target protein contains multiple isoforms or splice variants, resulting in inconsistent band sizes or the presence of multiple bands.
