hi,
i tried to purified mouse igM antibody by protein L and failed to do that. The concentration before and after is almost the same and almost nothing after elution.
I wonder if the problem is that there is some particles or salts in the sample and i should dialize the sample before loading the column.
Does anybody have some experience with that method?
thanks a lot for any advice
Wieslaw Swietnicki
Forget protein L and try proven solutions. Try IgM kit from Pierce (small scale) or Sigma column https://www.sigmaaldrich.com/catalog/product/sigma/ge17511001
1 votes
1 thanks
Maurice Ekpenyong
Hello Nevo, have you seen this article: Article Purification of antibodies using protein L-binding framework...
. I think your choice of protein L is in order. Binding of Protein L to immunoglobulins is restricted to those that contain kappa (κ) light chains (i.e. κ chain of the VL domain). In humans and mice, kappa (κ) light chains predominate. The remaining immunoglobulins have lambda (λ) light chains, which will not bind Protein L. Furthermore, Protein L is effective in binding only certain subtypes of kappa light chains. For example, it binds human VκI, VκIII and VκIV subtypes but does not bind the VκII subtype. Binding of mouse immunoglobulins is restricted to those having VκI light chains. The problem could be as a result of interferences. Your resins could also be a problem. You could use the sequential method. That is, start off with Protein L affinity chromatography, follow up with target-capture cation exchange chromatography and then with anion exchange chromatography. See the attached document for details.
0 votes
0 thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies
More Nevo Zeira's questions See All
Similar questions and discussions