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
Thank You kindly for any suggestions,
- Szymon Zietkiewicz
Intercollegiate Faculty of Biotechnology,
University of Gdansk