In article <351B64ED.20A6 at sars.uib.no>, litta.olsen at sars.uib.no wrote:
> For some time now I have been trying to make the 2-D system from
> Pharmacia work and have some trouble with this.
> I use premade Dry Strips and precast gels (8-18%)
> The first problem was that the front in the 2. dimension would move much
> faster on one side than on the other. This problem has been reduced, but
> still I see a smiling effect on one side, and since I do two parallel
> strips on one gel in order to compare them, this makes it rather
> I started out using the ph3-10 strips, but realised that they did not
> separate my proteines well and decided to try the pH4-7. I did the 2.
> dimension yesterday and it gave a strange pattern. It seems to me that
> all the proteines had "jumped" over about 1/3 of the strip.
> Approximately one third of the strip in the pH7 end was empty and it was
> not like it gradually dissapeared.There was a clear vertical split
> between the areas with an without proteines. I am sure this is due to
> the 1.dimension run, since both strips gave exactly the same pattern.
>> Has anyone an answer to this, then please contact me.
It could be a problem of precipitation of proteins during IEF.
Do you apply much protein or is there one protein especially
abundant? What kind of re-hydration buffer do you use?
I use routinely:
8 M urea, 2% CHAPS, 2% Pharmalytes, 11 mM DTT, which works better
in all cases than the buffer listed in the manual (using Triton).
The amount of CHAPS can be increased up to 4% for hydrophobic or
other "difficult" proteins. Also, in the sample buffer I use
4 % CHAPS routinely.
By the way, how did you solve the "smiling" problem in 2nd Dimension?
I have got the same problem and would appreciate any helpful advice.