Question (2330)
Question
After introducing my over-expression vector/virus into my cell line, I performed Western blot but the detected molecular weight differs from the expected size. Why is this and what troubleshooting steps should I take?
ABM community
Verified customer
Asked on Jan 29 2026
Answer
Differences between the observed and expected molecular weight are relatively common and can result from several factors, including alternative splicing, post-translational modifications, proteolytic processing, or the presence of fusion tags. In some cases, the antibody may also recognize non-specific proteins.
Basic troubleshooting steps include:
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Confirming the predicted protein size, including any tags or signal peptides.
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Verifying antibody specificity and reviewing the datasheet for known cross-reactivity.
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Checking whether the protein undergoes cleavage or modification in your cell type.
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Comparing expression to a positive control or reference sample.
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Confirming construct sequence and reading frame.
ABM Scientific Support
Answered on Jan 29 2026