Dear Dr.Kim,
It probably did work, but 5% FCS is a lot and probably exceeded the capacity of the Blue Sepharose gel for the sample that was used. When I have had a similar challange in the past, I used a bulk albumin removal method first. I sent an earlier ABRF communication with a bunch of suggestions (from experience), but unfortunately didn't keep a copy for myself. If you check the archives, tou will find useful hints. My communique was early last December. The following is all that I have remaining in my personal archive.
__________________________________
Dear Paul:
I read your message regarding BSA removal at the ABRF discussion list
and I would like to ask you for some further help.
I am trying to purify a scarce protein from a cell culture supernatant
containing 5% FCS. I have tried a couple of times a blue-sepharose
column and I havenĄt been able to remove more than a small fraction of
the total BSA content. I already confirmed my column is working using
comercial BSA fraction V from SIGMA. The SDS-PAGE migration pattern of
the retained BSA is different from that of the unretained so I am afraid
I wonĄt reach to solve my problem just improving my chromatography.
After I read your message I tried the pH precipitation at 4.8 with
sodium acetate but I didnĄt obtain any success. Among all the options
you give in your message this is the more likely to be usefull for us
because I know our protein doesnĄt precipitate until pH 3. I tried the
precipitation with the unbound fraction after blue-sepharose
chromatography being in Tris 25 mM pH 7.0 and also directly from the
culture supernatant and any of these two times obtained precipitation.
Is there any clue you can give me about this?
By the way, have you experienced your proteins migrate likely being of
higher molecular weights in SDS-PAGE in presence of this high BSA
content?
Thanks in advance,
Vivian
Vivian Huerta
Physical-Chemistry Department
Center for Genetic Engineering and Biotechnology
Havana, Cuba
_____________________________________________
Dear Vivian,
I am most concerned about the observation that precipitated BSA migrates differently than non-precipitated BSA on SDS-PAGE gels. It is also possible that upon final removal of BSA, that your scarce protein could become unstable and be lost on glass surfaces. If you could give me more information about your protein (pI, hydrophobicity, identity, post-translational modifications, size, metal binding character,...) it would be helpful. Is it an enzyme (like a peroxidase) that could conceivably modify BSA or bind to it some way? Are the two electrophoretic forms of BSA seen only after attempted pH precipitation?
If I had your problem, I would take a sample and split it into a dozen aliquots for study. One aliquot would be pH precipitated by the standard method, then the supernate concentrated to obtain the same or higher protein concentration and the precipitation attempted again (and so on until no further precipition occurs). Then, I would "play" with the sample trying alternative pHs (lower?) for further precipitation of the "new modified BSA", trying ice cold precipitation, trying precipitation in the presence of 2M NaCl, ethanol, or dialyzed against distilled water. If the new BSA species has a higher molecular weight, than repeated selective size exclusion with S-300 HR or some similar gel might be an option. The remaining sample aliquots can be used for exploring separation options in a different order (particularly if pH precipitation is promoting some form of BSA modification.
I know that this sounds like a lot of work, but without further info, I don't know what else to suggest. What happens with ion exchange or hydrophobic chromatography?
Good luck, hope some of this helps.
Sincerely, Paul Tressel
Paul Tressel
Senior Scientist
Hamilton Civic Hospitals Research Center
711 Concession St., Hamilton, Ont. L8V1C3, Canada
(905)527-2299 (ptressel@thrombosis.hhscr.org)
>>> "Helen Kim, Ph.D." <Helen.Kim@ccc.uab.edu> 02/16 5:42 PM >>>
Hi All,
Has anyone had experience removing albumin with Blue-sepharose? I
know in principle it can be done, and I've read lots of papers where people
blithely say they did with the blue sepharose. I recommended the
procedure to a grad student dealing with blob of albumin in his 2-D gels
of conditioned media. He bought some, tried it (I don't know exactly what
he did), and he says "it didn't work." He's very bright, though, so I trust
he went through reasonable efforts, but I thought I would check with you
all, since there's no point re-inventing the wheel.
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