Hi there,

IP is not compatible with denaturing condition as it is based on the recognition of an epitope by an antibody. The binding will be efficient if the antibody is under its native state only.

Dear Isadora,

can you provide more information about your protein and why you have to extract under denaturing conditions? And what exactly is your extraction buffer? And how do you denature your protein? In such experiments details matter a lot.

Cheers Markus

Pull down of ubiquitylated protein is often performed with an Histidine tag on your protein of interest (or on Ubiquitin) and with a Nickel- or Cobalt-based resin. In fact the biding HIS tag/Nickel happens also in denaturing conditions, when Immunoprecipitation is not available.

Hello, Markus

In fact I am trying to perform a in vivo ubiquitin assay in classic RIPA buffer. I have read in many papers that the IP is done with protein under denaturing conditions. So far I denatured it by boiling 5 minutes under 95 °C in the lysis buffer with 1% SDS and 1mM DTT, followed by dilution 1:5 in the lyses buffer containing NP40 or triton.

However, when I quantify the lysate I see a massive decrease in total protein concentration. Do you have a sugeestion?

Thanks, Davide, I will look into this possibility!

Dear Isadora,

can you please post the links to the protocols / publications? Also, please try to formulate your research question even more specifically, i.e. are you interested in a particular protein vs. overall ubiquitinylation? The experimental approach best suited to answer your question does of course depend a lot on your actual question. The better you can phrase that question the better responses you can get for the experimental approach to tackle it.

Cheers Markus