smori at opus.nmsu.edu
Fri May 27 13:15:30 EST 1994
Robert C. Colgrove (robin at alumni.caltech.edu) wrote:
: Ok, this is a pretty remedial question but I just noticed that
: practice in my lab seems to vary considerably from standard protocols.
: We transfer 20-200k proteins from 1-2mm 10-15% acrylamide gels onto
: nitrocellulose. We use those little biorad gel casters so out gels are
: about 8x6 cm. Now, all the printed protocols I see say to transfer for
: 1.5-2 hr at 0.5-1 milliamps per sq cm of gel. Everybody around here
: just does 2 hr at 100 mAmps no matter what the size gel. Does this matter?
: I was just running a 4x5 cm gel and started to get nervous.
: 20 mA (at 10 V according to the power supply reading) seemed pretty feeble
: compared with voltages we use fol _running_ the gel but the "lab standard"
: 100 mA seemed like overshooting by a huge amount. I wimped out and am running
: at 45mA x 1 hr in the standard tris/glycine/methanol buffer.
: Does anybody know what really matters here?
: thanks and sorry for the dumb question,
: robin colgrove
: division of infectious diseases
: harvard school of medicine
I do my transfers at 100 volts ( 40mAmps) at 4 C and I get good transfer after
1hr. Some of the high molecular stuff doesn't get transfer completely though,
but if your protein is in the mid-low MW this will give you a great transfer.
Also as loading buffer I use Pyronin Y which will transfer from the gel to
the NC and this allows you to determine the efficiency of your transfer.
Program in Molecular Biology
Dept. of Chemistry Box 3C
NMSU Las Cruces NM
|| "From The Land Of Enchantment"
More information about the Methods