Hope this small piece is helpful,
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403
On Thu, 13 May 1999, Helene Z. Hill, Ph.D. wrote:
> 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.
> 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) wa!
s 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.
> 5. So where did the actin go in the HPLC?
> How come proteins that were about 40 kD became 57 kD after HPLC?
> Why do the fractions that contain the coomassie stain appear to be empty of protein?
> 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.
>
> 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
>
>