Hi Kien- Are you basing extensive proteolysis (degradation) on an SDS-PAGE result alone? Are you monitoring the elution profile from Ni/NTA? Do you see numerous A280 peaks for your elution? Sample prep of membrane proteins for SDS-PAGE is very trial-and-error. Heating the samples may cause weird aggregation to occur. You often see a ladder effect and anomalous mobilities of membrane proteins on SDS-PAGE. This is clarified (normally) with a native gel and/or with analytical SEC. Now, I admit, this is normally for oligomeric membrane proteins- I'm not sure if yours is oligomeric or not. And this may very well not be your main problem because I would expect you would see some signal on your blot from your Ni/NTA flow through.
Hope this helps- Brad On Thu, Mar 19, 2009 at 7:53 PM, Kn Ly <kn...@auckland.ac.nz> wrote: > Hello everyone, > > I am expressing a 100 KDa eukaryotic membrane protein in E coli. The > protein > is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa. > > However, the protein get severely degraded so after putting through a > Ni-NTA > column, the protein came out with a lot of contaminant bands. I did a > western blot using antibody against his tag. The total cell lysate gave > signals in many bands. The flow through did not give any signal and the > eluted fraction again gave many band signals, indicating the protein got > degraded copiously even before purification. > > I used Roche protease inhibitor tablet and still got a lot of degradation. > Can anyone suggest a way to avoid the problem or a purification method so > that I can purify the intact protein while keeping away the unwanted > degraded fractions. > > Thanks heaps in advance. > > Kien >
