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[Maitreyi]

Hi! i am trying to purify a 125kD protein cloned in to pET32a. i do not get very good expression but it is sufficient for my work. i also get the protein in the soluble fraction inspite of its large size probably because of low expression. but this i have observed only when i digest the cells in micro amounts, that is 1.5ml. i use lysozyme and tritonx-100 to lyse my cells. off late i am trying to use larger amounts that is about 20ml of cells but i am not abole to digest the cells completely. as a result i have a significant amount lost in the pellet and i cannot aford this as the protein expression is already very low. is there any way to tackle the problem. as time goes by i want to use even larger amounts for digestion.

thanx in advance.

maitreyi

[anonymous]

Hi Maitreyi,

Is the protein you're trying to purify able to withstand ultra-sonification? If so, that's perhaps a better method to lyse more cells. Or perhaps a combination of chemical and physical lysis?

Kind regards,

Vincent Groenewold