PAGE of basic proteins
ds4 at st-andrews.ac.uk
Wed Jul 17 06:43:40 EST 1996
Hi Dave and all,
The mewthod we are using for native acid PAGE of basic proteins (or, in our
case, antibacterial peptides) is taken from Lehrer et al. (1991), J. Immunol. Methods 137, 167-173. Briefly, PAGE is carried out with continuous gels composed
of 6.4g urea, 9 ml distilled water, 0.8 ml 10% APS, 5.33 ml of a solution of
43.2% glacial acetic acid (AA) and 4% TEMED and 3.81 ml of acrylamide(60%)-
bis(1.6%). This suffices to pour 2 minigels for our BioRad mini protean II.
The gels are run with 5% glacial AA. Pre-run ca. 45 min at 150 V. The sample
buffer I am using (double strength) consists of 6 M urea, 10% glacial AA and
0.1% methyl green.
The electrodes are indeed reversed.
By the way, has anyone any ideas as to how to blot native gels? We must run
them native to make antibacterial overlays (see Lehrer et al. When the gel
is washed in phosphate buffer, then placed on a bacterial lawn for 1-3h,
clear zones will become apparent which correspond to the position of anti-
bacterial peptides or proteins). It would make our lives easier if these
gels could also be blotted. Would it be sufficient to soak them in transfer
buffer or would the methanol actually fix them and make transfer more diffi-
cult? Are there alternative protocols? Sorry if this sounds stupid, blotting
is another thing I am just getting into.
Gatty Marine Laboratory Fax: (0)1334-463443
University of St. Andrews e-mail: ds4 at st-and.ac.uk
St. Andrews Fife KY16 8LB
(Opinions expressed here are my own)
More information about the Methods