I have an organelle of interest captured by an antibody. I would like to elute the organelle for downstream analysis by LC-MS. We expressed a cMYC tag on a surface protein, then captured the organelle with anti-cMYC antibodies that were attached to magnetic dynabeads by avidin-biotin. I will only have a small amount of total protein, so I would like to minimize sample loss by avoiding sample handling as much as possible (e.g. fractionation, de-salting). From what I have read in various protocols, harsh elution conditions (e.g. SDS, 8M urea) will elute the antibody from the beads in addition to eluting the captured protein, which will contaminate the sample with antibody. The two soft elution conditions I have seen are high salt, which is undesirable because it will require de-salting, which I'd like to avoid; and 0.1 M Glycine*HCl pH 2.5. Glycine will not interfere with LC-MS analysis, but will interfere with BCA, making it impossible to quantify the protein and determine how much trypsin to add. Has anyone ever used straight unbuffered acid (e.g. formic or acetic) to elute an antibody? That could be easily neutralized and/or removed by speed vac.
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