Low/No IP Signal from Insufficient Target Expression

Symptom

IP experiment yields no detectable signal on western blot. Input lysate control shows weak or absent target protein bands, indicating expression below detection threshold.

1. 1 Target protein or interacting proteins expressed at levels below western blot detection limit
2. 2 Cell line or tissue model does not sufficiently express the target protein of interest
3. 3 Inappropriate cell line or tissue selection based on expression profiling data

1. 1 Use expression profiling tools (BioGPS, The Human Protein Atlas) and literature to verify target expression in chosen model
2. 2 Include known positive control from Control Treatments by Target table to confirm experimental setup
3. 3 Include input lysate control to verify target protein expression levels and antibody reactivity
4. 4 Select alternative cell lines or tissues with documented higher expression of target protein

Related Video (2)

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Western Blotting Protocol

"The Western Blotting Protocol is essential because detecting weak/absent IP signal relies on optimizing and correctly performing the downstream western blot readout."

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How CUT&RUN Profiles Chromatin | Cell Signaling Technology

"CUT&RUN is a chromatin immunoprecipitation-based method, illustrating IP experimental context and the importance of target abundance in pull-down assays."