I m working on cell culture and i need to analyse the set of proteins and their different additional groups at diffrent conditions and i dont know how to prepare sample for HPLC and LC-MS , i want to knw the additional groups added in my target protein compare to the control that is protein modification at treated group
https://www.researchgate.net/post/Sugar_preparation_for_HPLC
As a student at your university, please speak with the people who have experience with and run the HPLC and or LC-MS systems at your University. *Your teachers and the people who manage the analytical instrumentation are in a position best to serve your needs (not the web).
Dear, Sunilgowda, I think you have to do solvent extraction of the proteins which you are targetting, but keeping few things in mind. pH plays very crucial role in maintaining isoelectric point of the proteins, so that biological activity of the protein will remain intact. But, in that case, you must use some inert solvents like DCM which should not react with protein and functional groups. You can use equi-volume extraction and for method development prior to LC-MS, you have to optimize mobile phase composition using different composition of solvents and buffer. You have to extensively search the literature for previously reported protocols regarding the selection of mobile phase, flow rate, column and HPLC conditions, etc. for protein purification and elution. Further, you can use affinity chromatography followed by dialysis for protein purification.I hope it may help you.
Best regards
-Kshitiz
Dwivedi sir thank you for your information and i will go through it thank you
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