gmcstay

Hello Samuel,

it does sound like your HA tag is inaccessible to the antibody.  You could try different detergents to change conformation and complexes slightly.  You may also try inserting the HA tag in another region of the protein, maybe the other end of the polypeptide chain, or even insert it within the coding sequence.  

Gavin

Hello Xu Zhu,

quantifying activity of specific caspases is not as simple as it first seems.  The available substrates are not specific for each caspase.  The "specific" substrates for caspase-8 and caspase-9 are also cleaved very well by caspase-3.  In most cases you are simply measuring caspase-3 activity with these assays.  

There are some activity based probes available.  One is biotinylated-VAD-FMK, which is commercially available, and there are some derivatives made by the laboratory of Matthew Bogyo.  You can do a streptavidin pull-down and determine which caspase has been bound (indicating activation) by western blot.

I had some success with immunoprecipitating caspases and determining activity associated with the immunoprecipitated caspase.  I only did this in cytosolic extracts activated with cytochrome c and dATP to engage the apoptosome pathway.  I never tried it in cells I treated with an apoptotic stimuli.  

Here are the papers my comments are based on.

http://www.nature.co...l/4402260a.html
http://www.nature.co...ll/ncb1340.html
http://www.sciencedi...074552112000208
http://www.ncbi.nlm....les/PMC3365438/

Good luck and let me know if you have any more questions.

Gavin

Hello,

after the transfer, just keep the PVDF membrane dry, then when the antibody arrives, wet the the membrane with methanol, wash a few times in your western blotting wash buffer (TBS-Tween or similar) then block as you would be doing a normal immunoblot.  I do this all the time, and membranes can be stored indefinitely in this manner.

Gavin

Hello Florian,

I have been doing a very similar kind of thing using mitochondrial proteins that have been tagged with an epitope tag that can be eluted under native conditions.  However, these are proteins that are introduced into cells and not the endogenous proteins themselves.  You may be able to elute your protein with the peptide the antibody was raised against (if this is how the antibody was made).  

As for the amount of protein to load, when you have an IPed sample you purify the sample so much that the amount of protein loaded on to the gel is really not a problem.  I use 500ug of mitochondrial protein for my IP and when I do a western transfer of the gel I can see the two protein complexes stained by ponceau.  BN-PAGE gels can resolve 500ug of mitochondrial protein fairly well, but I would routinely use 250ug for sharper bands.  But an IP should not be a problem.  

Hope this helps you out in some way,

Gavin

Staurosporine is a very toxic chemical and induces apoptosis very quickly.  You can try shorter time points as you suggested, but also you can titrate to lower concentrations of staurosporine and check after 24 hours or shorter time periods.

Gavin