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.
Affinity Chromatography Help!?!