[Protein-analysis] Protein crosslinking with APDP?

Szymon Z. szymon at please.no.more.spam
Thu Nov 3 07:08:46 EST 2005


Hello,

I am attempting to perform some experiments that inwolve cross-linking with 
Pierce photoreactive bifunctional cross-linker APDP.
As I have no practice using that reagent, any advice would be most welcome.

I plan to label one protein via sulphydryl end of APDP and then use the 
labelled protein in a reaction. After that the glycerol gradient will be 
run, and  appropriate fractions will be flashed with light to activate the 
photoreactive group of APDP and cross-link that protein to whatever it has 
in proximity. I have read the papers provided by Pierce, but still have some 
questions, as follows:

 - Does APDP decompose thermally readily? Will it be possible to heat 
APDP-bound protein (that first one) for some 10 min, or will it destroy the 
photoreactive end (as I am affraid)? And how long can I keep APDP-bound 
protein sample before actual cross-linking with flashlight?

- How photosensitive it is? Do I have to worry about ambient light when 
labelling the first protein by sulphydryl?

- During the first reaction, sulphydryl labelling, without the light - can I 
have amines like TRIS in the buffer? I know they are to be removed before 
photoactivating the amino-specific, photoreactivable group; but will they be 
disturbing anything at the previous step?

- Is it a good idea to use a high-output photographic flashlight for 
cross-linking?  ISeems to me that it would give a possibility to precisely 
adjust amount of light by manually setting the discharge time, and 
additionally improve reproducibility -  while our hand-held UV source is 
quite weak, and it would be harder to manipulate with amount of energy 
irradiating sample.


Thank You kindly for any suggestions,

 - Szymon Zietkiewicz

Intercollegiate Faculty of Biotechnology,
University of Gdansk
Poland 



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