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I hope that one of you out there can figure this one out. Here are the =
facts:
1. Serum-free conditioned medium from cultured melanoma cells rescues =
irradiated and chemotherapy-treated cells from death (colony-forming =
ability). It was concentrated and separated by preparative zonal =
electrophoresis (PZE).
2. Neutralizing antiserum (As50) was obtained using activity fractions =
from PZE. On western, the antiserum reacts with 2 proteins of about 40 =
kD in the fractions that have activity in the bioassay. I call the 2 =
proteins upper and lower.=20
3. On MALDI-MS, lower could not be identified and upper appeared to be =
actin but we now know it is contaminated with actin.
4. Upper and lower were excised from coomassie-stained prep gels, =
electroeluted, concentrated and treated with BioBeads to get rid of =
remaining SDS and added PBS (no Ca or Mg). Added anti-Actin (Sigma) at =
1/400 to upper and incubated at 4 deg over the weekend. Centrifuged 30 =
min in microfuge. SDS-PAGE and western showed that pptn was incomplete. =
Upper SN contains upper protein, actin and actin-antibody complex. =
Upper SN, lower and Sigma actin were run on HPLC in 0.1% THF and =
acetonitrile 0-60% at 1 ml/min. Actin elutes at 7-8 minutes. There =
were 3 bands for upper SN, 5-7, 43-44 and 64-66 minutes. The last was =
intensely blue. There were 3 bands for lower, 2 at 5-7 min which I =
pooled and one at 61-63 min which was intensely blue. We assumed that =
the proteins we were purifying would be where the blue was. The peak =
samples were concentrated by speed-vac, resuspended in 0.0625M tris pH =
6.8 and 20% of each (10% of the blue fractions since these were the =
biggest peaks) was run on SDS-PAGE for westerns along with actin. Actin =
ran at about 40 kD and was the only lane that reacted with actin Ab. =
Upper fractions 5-7 and 43-44 had strong reactions with As50 but the MW =
of both was ~57 kD (remember it started at ~40!). Lower fractions 5-7 =
ran funny but was slaunchwise between 40 and 57 kD, a repeat ran at 57 =
kD. The blue fractions did not react with either antibody. =20
5. So where did the actin go in the HPLC? =20
How come proteins that were about 40 kD became 57 kD after HPLC? =20
Why do the fractions that contain the coomassie stain appear to be =
empty of protein? =20
Did the HPLC reagents strip the dye from the proteins?
Can anyone tell me a better way to get the actin out of upper?
Does anyone know where to expect the immunoglobin or the complex to =
come off in the HPLC?
You all have been wonderfully helpful before. I have learned a lot from =
your e-mail conversations with each other. I am learning all of this =
the hard way and these latest results really have me baffled. Any help =
I can get would be greatly appreciated. =20
Lanie Hill
Helene Z. Hill, Ph.D.
Professor of Radiology
NJ Medical School
Newark, NJ 07103-2714
Tel:973-972-3421
Fax:973-972-5592
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