The affinity beads was obtained from Promega and commonly used for GST-fusion proteins. I am trying to use this beads to enrich native GSTs in human plasma but right now I cannot get satisfied results, because GSTs are very low abundant in plasma (5~40 micrograms per liter) and there is non-specific binding. I have tried different conditions to have better binding efficiency. The enrichment procedures are basically demonstrated as following.
200 ul plasma was incubated with GSH magnetic beads at room temperature for 2 hours and then the supernatant was discarded. After three times washing of the beads to remove non-specific binding proteins, the bind GSTs were reduced with DTT (10mM) and then alkylated with IAM (55mM). After these steps, the beads was incubated with trypsin at 37℃ for overnight digestion. The digested peptides were delivered to LC-MS analysis.
So does anyone have idea about this issue that how to enrich more GSTs? Thanks very much.
Is it possible that reduced glutathione concentration (GSH) is high in plasma and competing for binding to GSTs which are already at very low concentration as you mentioned?
Actually I did not think about this point before, because GSH is easy to oxidize. Besides, I checked some publications and the concentration of GSH in healthy plasma is around 15 nmol/mL (see the following link) and we are recommended to use 10 mM GSH to elute the binding GST. But in my experiment, there is no elution but digestion on the beads directly.
https://www.ncbi.nlm.nih.gov/pubmed/16499058
Dear Cornelius,
Thanks for your answering.
Yes, what I want to capture is trace amount in plasma, but according to previous experiments, several members could be identified by LC-MS/MS. I want to know is there any way to improve the binding efficiency to get more and stable identifications, so we can quantify those GSTs individually by LC-MS/MS without antibody.
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