Hi everyone,

I'm currently working on a ChIP-seq experiment and encountering an issue I hope someone can help me troubleshoot.

My experimental workflow overview is as follows:

• Cloned a His-tagged protein into the pHERD20T plasmid, confirmed by Sanger sequencing.

• Expressed in Pseudomonas aeruginosa PAO1 using the arabinose-inducible system.

• Lysed cells and performed IP using Dynabeads Protein A and Anti-His antibody.

• Followed with reverse crosslinking, proteinase K treatment, although DNA purification, and sequencing are still to be done.

To validate the workflow, I ran SDS-PAGE and Western blot after IP.

SDS-PAGE and Western Blot Observations:

• No enrichment observed in the absence of anti-His antibody. So added anti-his Ab for IP with the kit.

• After IP, SDS-PAGE showed a band slightly above 11 kDa (expected size), but no corresponding band appeared in the Western blot.

• Could this SDS-PAGE band represent the protein complexed with DNA?

In the ChIP eluate, I observed two bands—possibly one from the antibody and one from a DNA-protein-antibody complex.

I proceeded with reverse crosslinking and ran a ChIP validation blot comparing TDC (total lysate) and ChIP elution samples across three stages: before and after reverse crosslinking, and after proteinase K treatment. I expected that reverse crosslinking would release my His-tagged protein from the DNA complex, allowing it to appear at its expected size (~11 kDa) on the blot. However, the protein band was absent in the ChIP elution, and the same two bands observed in earlier blots persisted. No band was detected in any TDC sample either.

The gels/blots have been attached for reference. The samples include:

• Pro1, a purified His-tagged protein from E. coli BL21, to confirm expected band size.
• TDC1–3: Before/after reverse crosslinking and proteinase K.
• ChIP E1–E3: Same stages for ChIP eluate.

Questions

1. How can I distinguish between lack of expression and failed IP?

2. What could explain the absence of band in TDC?

3. What controls or validation steps can confirm protein expression before IP?

4. How can I optimize reverse crosslinking to release protein from complexes?

5. Has anyone dealt with protein-DNA retention or antibody contamination in ChIP eluates?

6. I also encountered gel running issues (smiling effect)—any tips to avoid this?

Any suggestions, protocols, or shared experiences related to ChIP-seq, DNA recovery, or protein detection would be greatly appreciated!

Regards,

Thank you!