2D-PAGE PROTEIN ANALYSIS - SECRETED PROTEIN DESALTING (Apr/13/2007 )

Hi all!

I am going to start my work on 2D-PAGE analysis of secreted protein of sertoli cells. but the problem is that i dont know how to desalt my proteins and concentrate them. i want my proteins to be free from salts before running in first dimension. can anybody help?

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you can desalt while concentrating (called "diafiltration"). concentrate then dilute with salt-free buffer and concentrate again. repeat as necessary.

or

you can concentrate then dialyze (various apparati available for various volumes).

or

you can dialyze then concentrate.

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Following an elder advice I would like also to suggest the following : Hitrap desalting 1 or 5ml depends on your sample volume + microcon with Mw depends on Mw of your protein . This steps may rotate

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hi

thanks for your reply, i will try to do the same. in case, u happen to have any protocol 4 the same plz. send me, if possible 4 u.
Am i supposed to desalt cell lysate proteins also be4 running them in 2D-PAGE

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no special protocol, just follow the directions for use of the concentrator that you use and for the dialysis membrane or desalting column that you select.

you can desalt the lysate if you wish. it depends on what you use to lyse that determines if you have to desalt. just remember to clarify it before applying to the gel.

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