Howdy: Here are my experiences with IEF transfers. From native IEF, we have
never had problems transfering proteins. Word of caution, proteins are readily
mobile from gel to membrane, so be very careful to not move the sandwich
assembly - leads to smearing!!. I routinely use CAPS, pH 11.0 + 5% methanol.
Methanol is not needed for NC transfers, but helps in PVDF transfers. For
urea/non-ionic detergent IEF, we use pretty much the same buffer system, but I
let the gel equilibrate for about 10 to 15 mins in CAPS transfer buffer. At pH
11.0 most proteins will transfer to the anode side. However, if the proteins
have a very alkaline pI, transfer efficiency will be compromized. In these
instances, it may be better to use a less basic transfer buffer and use Towbin's
or even MES, pH 6.0 and try transfrering to the cathode side. In any event, gel
equlibration for urea-IEF gels will improve transfer. I take it these
transfers are from vertical IEF gels and not from plastic backed strips. All of
my conditions are for vertical IEF in slab gels. That's about 2 cents worth,
bye, Gautam
Gautam Sarath
Research Associate Professor, Biochemistry
& Manager, Protein Core Facility, Center for Biotechnology
N-226, The Beadle Center, 19th and Vine Street
University of Nebraska, Lincoln
Lincoln, NE 68588-0664
Tel: 1-402-472-2928; Fax: 1-402-472-7842
http://www.biotech.unl.edu/Proteins/index.html
http://www.pigment.unl.edu/dept/sarath/sarath.html
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