Try PLA (proximity ligation assay) for assessing possible protein-protein interaction on tissue sections. Kit is available with sigma.

Either your protein natively forms cross-links (which is a possibility) or your SDS gel sample buffer is not stringent enough to break up its binding to other ligands.

Have you tried increasing the stringency of the sample buffer (e.g. addition of urea)?

Thank you Dr. Rahul. I will give it a shot and see if that can be worked with my samples.

Dr. Holger I have a doubt.

First of all thank you for your suggestion. I have not tried adding urea to my sample buffer but may try out that option too. However, as per your saying the possibility of having self cross-linked can happen in the formaldehyde samples and this should not happen in my control where only 1XPBS was used as cross-linker.

Hi Mayank,

I don't know where your protein is located: intra- or extracellularly.

Particularly extracellular proteins can be cross-linked by several mechanisms: heterologous disulphide bonds, ROS cross-linking, etc.

By using a stronger / more stringent sample buffer it should be possible to exclude non cross-linked interactions, which can be quite strong. By adding 6 - 8 M urea to both your gel sample buffer and possibly also to your whole gel (self-cast gels), you can dissolve many non-covalent complexes that may resist denaturation under less harsh conditions.