Good Day ABRFers,
Thank you all so much for your responses to my question and excellent
suggestions regarding the DigestPro. Indeed, it was a problem with the
black inner needle of the liquid delivery system. I spoke with Dean Stott
at LC Packings last week and over the phone, he helped me adjust the black
inner needle so that it extends 6-8 mm from the stainless steel outer
needle. Fixing this and then reseting the z-height solved the problem with
the liquid spilling over the top of the tubes. Great!!
Now, we have another problem. It appears that the proteins in the gel
bands are not being digested anymore. We always run 5, 10 and 20 pmol BSA
samples as standards protein for digestion. BSA is not being digested at
all. We analyze the digests by MALDI-TOF MS (our MALDI-TOF ms is working
fine). Other proteins bands are not being digested either. The only clue
I have for this dilema is that there appears to be residual iodoacetamide
in the collection tubes because the samples turn yellow when they are dried
in a speed vac. We have never seen this before. My working theory is that
residual iodoacetamide is alkylating the trypsin and inactivating it. I
have checked liquid delivery and expulsion. It is working fine as far as I
can tell. It is unclear how the iodoacetamide could linger.
I would appreciate any ideas you may have. Thanks in advance everyone.
-Chris-
(I think that at this point I am supposed to say something like "Weather
Inconsequential." Well, here goes)
Weather Inconsequential: San Diego is in the midst of a "Santa Anna"
weather pattern. Warm, dry air from the deserts has cleared the skies and
raised the temps. It has been beautiful, warm and sunny today and
yesterday.
***********************************
Christian R. Lombardo, PhD
Director, Molecular Analysis Facility
The Burnham Institute
10901 North Torrey Pines Road
La Jolla, CA 92037
858-646-3108
crl@burnham-inst.org
***********************************
This archive was generated by hypermail 2b29 : Wed Feb 09 2000 - 10:35:56 EST