No Protein Detected: Lysis Buffer Incompatibility

Symptom

Target protein not detected in IP despite using sufficient antibody and confirmed protein expression. Antibody may fail to recognize target due to protein denaturation or improper folding.

1. 1 Denaturing lysis buffer used with antibody that only recognizes native protein epitopes
2. 2 Non-denaturing lysis buffer used when antibody requires denatured protein for recognition
3. 3 Lysis buffer contains detergents or agents that disrupt antibody-antigen binding
4. 4 Inappropriate buffer composition causes protein aggregation or precipitation

1. 1 Check antibody datasheet for recommended applications: IP typically requires antibodies that recognize native proteins
2. 2 Use non-denaturing lysis buffer (e.g., 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, protease inhibitors) for native IP
3. 3 Avoid strong ionic detergents (SDS >0.1%, sarkosyl) or high urea concentrations that denature proteins
4. 4 Verify lysis buffer compatibility with antibody isotype and adjust detergent type/concentration accordingly

Related Video (2)

Cell Signaling Technology ★ 72

Western Blotting Protocol

"Western blot protocol demonstrates protein sample handling and lysis context closely linked to IP workflows, helping understand native vs denaturing buffer pitfalls."

Cell Signaling Technology ★ 68

How CUT&RUN Profiles Chromatin | Cell Signaling Technology

"CUT&RUN is a chromatin immunoprecipitation-based assay, so it shows IP/antibody-based context and how buffer and antibody compatibility matter."