western blot - western blot (May/27/2009 )
could you guve me a protocol for gradient gel?
tnks
do you want the complete gel formulation or are you just asking how to pour a gradient?
i think..there are commercial gel 3.5-5% gradient gel with a 8-0M urea gradient. is it true??
than...is possible and usefull trypsinize the protein and than see the difference on smaller fragments..what do you think??
distazio on Jun 4 2009, 06:02 AM said:
i haven't seen them, but i haven't really looked.
are the proteins related? will you see cross reactivity with the same antibody?
if not then you could stain, strip and restain if the proteins are in the same lane or cut the membrane and stain separately if the proteins are in different lanes.
fragmenting and then staining seems to be overkill but you could do it if you know the fragment pattern for each protein. keep in mind that the antibody will only detect the fragments that contain the epitope(s) that were used to inoculate.
tnks a lot.
but i suppose that is not easy to do. i'm working in a genetic lab and we haven't a great experience in proteins.
but for me is important to see the two myosin protein with 3 KDa of difference in my Knock-in ES cells. but i don't know which is the best and simple system to use.
tnks very much.
mariateresa from italy
i made an error in my description of the gel we used to separate the myosin isoforms (forgive me, we published in 1983 and i haven't used it since).
the gel was 3.2-5% acrylamide with a 0-8M urea gradient. the stack was 2.8%.
i will pm you with the paper and two supporting papers.
thanks you are really nice! i'll ready the papers. thank u.
i have another question: is it possible to obtain a specific protein cleavage?? and how??
mt
you can get a sort of specific cleavage. proteases will cleave at specific residues. but, you usually find several cleavage sites on your proteins.
in some cases you can introduce a specific site that is unique to a specific protease (eg thrombin) and is not otherwise found in your protein.