| |||||||||
| Login :: Register :: Search forums :: Top Users :: Reagent |
|
Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
|
help
Posted by: Prakasha Gowda (IP Hidden, Unregistered user, )
Date: April 20, 2005 12:36PM
Hi
Currently I have been using Iodinated cross linker to cross link IRBC surface proteins (plasmodium falcifarum infected red blood cells). After crosslink i will extract proteins with 2% SDS in PBS containing protease inhibitors. The extract shows about 100000 CPM in 30ul and I put half of this count on 4-15% acrylamide gel and run at constant voltage. Dried the gel and exposed for 7 days at room temperature (autoradiography) but I did not see any band. Some times most of the sample stays in the gel well. Would you please suggest me why I am not looking any band if I use 50000 CPM? Thank you. Sincerely, Prakasha Gowda Department of Biochemistry and Molecular Biology Pennsylvania state University College of Medicine 500 University drive Hershey, PA 17033 Phone work: 717-531-3523 Hershey
Re: help
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 20, 2005 12:52PM
Hi,
Is this cross linker homo or heterobifunctional and is it water soluble or insoluble? it seems that your protein ccomplex is huge and is not migrating through the gel and most probly is staying in the wells. if this the case you need to reduce your cross liker concentration used, one way to go around this is to test for the optimum cross linker conctration at first by doing a sort of cross linker increasing concentration curve )start at low conc and increase two folds) and thus you can obtimize your cross linker concentration used for this experiment. There is a cross linker guide that I have altered from Pierce is posted at [www.biolynx.ca], Good Luck!
|
We are moving ... Please post to our new community forums
|
|
||||||||