Your guess about solubility of membrane proteins in CHAPS is a good
one. You may be having problems with large complexes or aggregates that may or
may not migrate during IEF and are likely transferred inefficiently. After
spending a couple of years attempting to use milder conditions, I finally gave
up and tried the following, which seems to work well if done properly.
I solubilize the sample in as small a volume of 2% SDS as possible, with
boiling, if necessary (almost always necessary with my membrane proteins).
After cooling, I displace the SDS from the proteins with 5-8-fold excess of
nonionic detergent in the IEF loading buffer and run immediately. When the
IEF dimension is completed, I soak the IEF gel in SDS PAGE sample buffer for
20 min prior to running the second dimension.
This works reliably well; there are usually no "lanes" of proteins representing
aggregated complexes in the first dimension. Note on transfer of membrane
proteins to blots: Be sure to use the appropriate transfer conditions, i.e.,
longer transfer, higher voltage, 0.1% SDS and perhaps 1% methanol in the
transfer buffer.
Good luck!
______________________________ Reply Separator _________________________________
Subject: IEF efficiency
Author: Amanda Hall <mbah@medicine.newcastle.edu.au> at SMTPMED
Date: 2/22/98 9:28 PM
Hi everyone,
Back in January Judith Airey asked a question about efficiency of IEF gels.
I am having a similar problem and I missed the responses so I hope you
don't mind me asking a similar question. I am a beginner with 2D's and
would appeciate any help.
As with Judith I am finding that the amount of protein present at the end
of the 2nd dimension run is much less than expected. I am using the
Pharmacia IPG strips for the 1st dimension run.
My sample is a mixture of membrane proteins in CHAPS detergent. The sample
has protease inhibitors present so proteolysis shouldn't play a major part.
Could the solubility of the membrane proteins be the problem, perhaps with
the protein precipitating out during the 1st dimension run? If this is a
potential problem has anyone got any useful suggestions for increasing the
solubility of the sample, or any ways to improve efficiency generally.
Any comments would be gratefully received
Thank you very much
Amanda HAll
Amanda Hall
Professional Officer
Newcastle Protein
University of Newcastle
ph 02 4921 7299
email: mbah@medicine.newcastle.edu.au