Author Topic: Tris-Acetate gels for separation of high MW proteins and western blotting  (Read 5306 times)

EmNZ

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Hi, I am trying to resolve a GFP tagged GPCR which migrates as 3 bands representing different glycosylated forms between ~90-130kDa following transfer from a 7% Tris-Glycine gel. To get sufficient separation of these 3 bands I needed to run the 75kDa marker to the bottom of the gel, and in the process I would have lost the ~65kDa unglycosylated monomer which I would also like to see on my blot. I need to get accurate size estimates of the 3 glycosylated bands as this is really important to my model. I am considering trying a Tris-Acetate 3-8% gradient gel (or similar) to separate the ~90-130kDa bands as much as possible while retaining the ~65kDa band, prior to transfer onto PVDF. These gels are commercially available but I cannot find any independent recipes/protocols online. I am concerned that the commercial gels seem to all lack SDS and how this might affect migration of my proteins. Does anyone have any experience/advice to offer me? I would be enormously grateful as the last part of my PhD hangs on this result, many thanks in advance  :)

ElkeKS

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Re: Tris-Acetate gels for separation of high MW proteins and western blotting
« Reply #1 on: September 29, 2011, 05:54:44 AM »
Hi EmZ,
you should not worry about gels without SDS. In 2D gels, we often omit the SDS in the casting because experience shows that it migrates out towards the bottom anyway before your sample moves in. It is more relevant to have sufficient SDS in the sample buffer and the running buffer.
Hope that helps,
Elke