I observed a broadening of an elution peak in Gel filteration and my target protein elutes within this broaden peak. I am wondering about the possible cause of it. literature mostly says it happens because of poor column packing, however that's not the possible cause in my case. hwoever, i do belive it may be becuase of presence of various protein conformation, which were not well-seperated in gel filteration. i even performed a negative stain EM, it showed the seperation was not optimal. however, howeve, i do not understand how it can be possible because gel filteration is supposed to seperate proteins based on their hydrodyanamic radius?
Any suggests or explaination or literature would be apperciated.
Peak broadening in gel filtration chromatography can be influenced by several factors besides poor column packing. Since your column packing is not the issue, consider the following possibilities:
dear Meenal when you have time kindly have a look on word file which i attached.any confusion or question feel free to ask
best regards
actually i did a work like you (10 years before) when i was doing MPHIL from UVAS Lahore. but i lost my protein many times during purifying process like gell filtration etc . my Professor helped me alot Prof. Dr Tayyab(PHd from PU) and Prof. DR Atif Hanif helped me to do SDS-Page. it is really difficult and time consuming. i wish you will get success. don't worry. @
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