Posted 11 February 2011 - 07:23 AM
If I choose the wrong acrylamide concentration for my SDS-PAGE is it possible that I don't get my protein after incubating with my antibody?
In other words it is clear for me that a good separation is important to be sure that the protein I see on my blot is that I'm looking for, but do you think if I'm running a 18% gel and therefore the proteins do not separate well, it could be possible that I don't detect the one I'm interested in?
THanks in advacne
Posted 11 February 2011 - 11:41 AM
Posted 11 February 2011 - 09:26 PM
Also, see attached document for the concentrations to use for effective separation (Roche Lab FAQs, pg 92).
Posted 14 February 2011 - 05:43 AM
The problem that I have is related to the detection of small proteins (about 4 and 17 KDa) that I'm not sure are present in my sample and that so far I was not able to detect using a standard western method. I checked the forum and I found some suggestions to follow but I wanted just to understand if the run could be the cause of my failures.
Posted 14 February 2011 - 02:55 PM
Posted 15 February 2011 - 03:41 AM
With proteins of that size it is very likely that they are blowing through the membrane on transfer - what membrane type are you using and how long are you transferring for?
I started working with a 45 um nitrocellulose membrane and I blotted for about 30' at 100 V (wet). Reading the forum I'm pretty sure this are not the right conditions. Which do you think should be the best?
Posted 15 February 2011 - 01:13 PM
Posted 15 February 2011 - 01:26 PM
i would also use a tris-tricine gel instead of tris-glycine. it is better for separating low molecular weight proteins and peptides.
Edited by mdfenko, 15 February 2011 - 01:27 PM.
genius does what it must
i do what i get paid to do
Posted 17 February 2011 - 03:19 PM