Protein Purification Techniques:
Hi, I tried to purify Ig light chains (LC) from MM or AL patients' urine with slight proteinuria, that means there is still albumin in there, messing up my experiment. I tried virtually everything by now, dialysis and SEC do not work, the MW is too similar, old style fractionated precipitation, also nope. Special chromatographic media to deplete albumin? That all has a to low binding capacity. Antibody-Affinitychromatography specific for the LC? Also useless, the Albumin sticks so strong to the LC that it comes down from the column in the end. I also talked to a GE biochromatography expert, but she had not much to add to my attempts. I don't know if the Albumin binds via Disulfid-bridges to the LC, that would be horrible, because as soon as the LC is reduced from dimers to monomers it starts aggregation as hell. I would be thankful for every method that worked or even didn't work by now.
Dear Kathrin,
Protein G or A binds IgG very strongly, in my hands 2M NaCl was not destroying this interaction. For your purpose I would suggest to think about protein L. In the paper below you have written, but not shown... it is binding LC even under reducing conditions:
http://www.jbc.org/content/267/4/2234.short
Keep fingers cross - Jan
Hi Kathrin, you could try using Blue Sepharose to bind the albumin:
https://www.gelifesciences.co.jp/catalog/pdf/18106075.pdf
If that doesn't work than you can take advantage of the fact that serum albumin is one of the few plasma proteins that has a free thiol group (S-H).
You can incubate your sample with biotin maleimide:
http://www.sigmaaldrich.com/catalog/product/sigma/b1267?lang=en®ion=US
and remove it with a streptavidin resin.
There will be problem using protein-A/G since they tend to bind intact IgG not light chain or heavy chain (that is why we use protein-A-HPR for IP to avoid bands for heavy chain and light chain). Protein L will be the reasonable choice.
Thank you Guys for your ideas:
Protein L unfortunately only binds to kappa subtype light chains and I have lambdas, too. I tried something similar from GE LambdaFabSelect, but albumin stuck to the light chain even under the slight basic washing steps.
I tried the Blue Sepharose HP Columns from GE, but their capacity was way too low. The light chains are mainly abundant as dimers via disulfid-bridges and monomers on non-reducing SDS-PAGE, but form one higher MW band in native PAGE, the light chains could have free thiol groups, but the idea seam interesting enough to try it.
Protein-A/G is not binding light chains, so I considered it for depletion of Immunoglobulin traces in m samples. It worked nice and I consider it as helpful in kidney impaired patients, but only, if I can get rid of Albumin first.
need a method to break the strong binding between the light chains and the albumin first before I can do any affinity purification and I begin to believe the binding might be via the disulfide bridges.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques