Question (2322)
Question
I transfected cells with a non-viral CRISPR KO plasmid and see GFP, but my Western blot shows no reduction in protein expression. Why?
ABM community
Verified customer
Asked on Jan 16 2026
Answer
This is a common issue and does not necessarily indicate the KO failed.
Common causes include:
(1) KO assessment performed too early
GFP confirms transfection but does not guarantee functional KO at early time points. With transient systems, knockout efficiency may be limited at 24–48 hours. We recommend waiting 72–96 hours post-transfection before assessing KO outcomes. If possible, proceed with clonal isolation for the most reliable KO validation.
GFP confirms transfection but does not guarantee functional KO at early time points. With transient systems, knockout efficiency may be limited at 24–48 hours. We recommend waiting 72–96 hours post-transfection before assessing KO outcomes. If possible, proceed with clonal isolation for the most reliable KO validation.
(2) Protein/mRNA stability (long half-life)
Many proteins (and transcripts) have long half-lives, so residual signal may persist even after successful editing—especially in a pooled population where editing is heterogeneous.
Many proteins (and transcripts) have long half-lives, so residual signal may persist even after successful editing—especially in a pooled population where editing is heterogeneous.
Recommended KO validation workflow:
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Enrich GFP⁺ cells (FACS recommended if available)
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Extract genomic DNA from pooled cells
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PCR amplify the target region
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Perform Sanger sequencing and analyze indel frequency using ICE or TIDE
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Proceed with single-cell cloning and validate KO clones using gDNA sequencing ± RT-qPCR / western blot / functional assay
ABM Scientific Support
Answered on Jan 16 2026