Hi there,
I have lysed my E. coli culture using PopCulture reagent from Millipore.
Now I have to quantify a protein, and I want to use LC/MS/MS. I am aware that some detergents (eg SDS) will interfere with LC/MS/MS measures (when not killing the column); I was wondering whether anyone has tried before to run LC/MS/MS after PopCulture Reagent and whether there are protocols in place to clean the sample before tripsination.
Many thanks,
Miche
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1 reply to this topic
#1
Posted 20 May 2012 - 07:58 PM
#2
Posted 22 May 2012 - 05:30 AM
I don't have experience with this particular reagent but we use iTraq and our own cell lysis formulations, which contain surfactants. Techniques that we routinely use in our laboratory for removing surfactants include:
Something else to consider... Is the Popculture reagent compatible with tryptic digestion? Make sure the pH is buffered properly and that the detergent concentration isn't too high. A relatively low detergent concentration won't low digestion efficiency (in fact it could increase the efficiency), but extremely high surfactant concentration could inhibit digestion.
- Acetone/Acetonitrile precipitation
- Cation Exchange chromatographic techniques (disposable SCX spintip for example)
- Centrifugal Ultrafiltration and/or dialysis
- HPLC
Something else to consider... Is the Popculture reagent compatible with tryptic digestion? Make sure the pH is buffered properly and that the detergent concentration isn't too high. A relatively low detergent concentration won't low digestion efficiency (in fact it could increase the efficiency), but extremely high surfactant concentration could inhibit digestion.
- Arun Kumaran likes this
Lab Technician at Protea Biosciences