Affinity Chromatography Help!?!

2 posts / 0 new
Last post
alanine
alanine's picture
Affinity Chromatography Help!?!

 Hello,
I'm trying to separate lipoproteins using concanavalin A, for some work I'm doing. Briefly, I'm adding my sample to con A - sepharose in a 5 ml tube, and then recovering the non-bound fraction by centrifugation (after mixing). Then washes, collect the bound fraction ...
My problem is in trying to quantify the dilutional effects in order to calculate recovery. I thought of including a marker in the sample buffer to derive a dilution factor. I've tried urea, creatinine, glucose, and amylase. The results make no sense. Applying a factor I'm getting recoveries of about 110 %, without about 90 - 95 %.
I'm sure my question is going to highlight my school boy ignorance about the principles of the techniques, but I would really appreciate any thoughts or advice.
Thanks,
Steve

R Bishop
R Bishop's picture
Hi Steve,

Hi Steve,
Welcome to Scientist Solutions.  I've done a good bit of lipoprotein isolation over the years, but Ive never used ConA columns to separate them. Typically ultracentrifugation and heparin sepharose.  Many times I had to back calculate recovery or better stil percent purification.  Still it seems to me yours is conceptual problem.
I might need some more information from you, but here's my stab at your problem.
First?  you are adding what to the ConA? Serum or a mixture of lipoproteins?
You are starting with X volume of a mixture, lets say 1ml that is a given concentration 1mg/ml for example.
You recover Y mgs/ml in a set volume lets say 0.1mg/ml in 5mls thus creating the dilution effect you are speaking of.
In the end you recovered 0.05mg of lipoproteins (as measured by apolipoprotein concentration). Therefore your percent purification is  20-fold and the percent recovery is 5% of the original mass.
You dont have to worry about the washes since they dont affect the bound mass of lipoproteins. the same hold true in your unbound fraction post-centrifugation, you recover a concentration of lipos in a volume of buffer.  The dilution factor is the volume of buffer.
When i get confused on these types of questions or want to check myself, I use microcon concentrators to concentrate my lipoproteins back to the original starting volume (e.g., pre-ConA) and recheck the mass. Not necessary but useful.
I could be totally answering the wrong problem for you, but thats my best guess based off your description.  The same thing should have occured with glucose (ConA binds sugars right?). Your dilution factor is merely the recovery volume x the concentration of glucose bound. If Im way off shoot me some more info and I'll get back to you in a few.