Hello all,
We have been running some samples of protein on PVDF for a customer,
and have had a very distressing problem with disappearing amino
acids, including norleucine, which we add as an internal standard.
Amounts go down to 20% of what they should be. We haven't seen this
before with extraction off of PVDF---the norleucine was fine, even if
there wasn't enough protein to quantitate on the membrane. We run
vapor phase hydrolysis, with 6N HCl, 1% phenol, and five grains of
sodium sulfite. Hydrolysis is at 110 degrees, for 20 hours. We dry
samples for 10 minutes, then extract three times with 40% ACN, 0.5%
TFA, dry the extract, bring it up in Na-S sample buffer, and run on a
Beckman 6300 with sodium buffers.
I ran a 10-15% polyacrylamide gel (250mM Tris-HCl (pH 8.8), 0.1% SDS) at
low voltage (appx. 45V) overnight and throughout next day until bands as
spread out as much as possible.
The information from our customer about transfer follows. She used
Immobilon-P for the membrane.
I transfered the gel to Millipore Immobilon-P PVDF membrane by wet transfer
at low voltage (appx. 9V) overnight in buffer containing 25mM Tris-OAc (pH
8.8), 0.19M glycine, and 0.01% SDS.
After taking the transfer down, I stained the membrane in Coomassie for
5-10 minutes, then detstained (10% acetic acid, 10% methanol), changing the
destain several times. Then, I washed at least 4 times with fresh Milli-Q
water, one of those washes went overnight. I air dried the membrane,
resting on Saran Wrap, then wrapped the membrane in Saran Wrap while
preparing to cut the bands out.
I placed the membrane on a piece of glass cleaned with soap and water,
followed by ethanol. I cut each band out in a hexagon-ish shape with a
fresh razor blade, manipulating the band with tweasers, washed both with
ethanol frequently. I wrapped each band in Saran Wrap, which I had cut
with scissors I also washed in ethanol, wrapped the bands up and taped to a
piece of paper and sent to you.
We are also looking at partial injection failure, with percentages
being off because of low amounts, but 1) All the other samples were
fine 2) This has happened twice with these samples.
Anyone have any ideas?
thanks!
-laurey
Weather Inconsequential: The sun just broke out on what has been a
grey day. Too bad it can't rain instead of just threatening!
Laurey Steinke
Protein Structure Core Facility
University of Nebraska Medical Center
Omaha Nebraska, 68198-4525
Phone (402) 559-6647
FAX (402) 559-6650
lsteinke@molbio.unmc.edu
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