After preparing the cell lysate and after protein quantification, if i don't have any lysis buffer left, can I use water (+protease inhibitors) to prepare the protein sample for loading with the X4 loading sample buffer instead of using the Lysis buffer to dilute the loading buffer to X1?
In electrophoresis, you want the concentration of extraneous salts to be as low as possible. Therefore, it is best to use water for dilution. The pH is fixed by the SDS-sample buffer.
Dear Itai
I suggest to you to use a buffer as Tris pH=8 or PBS , because in water the pH is not regulated, ionic strength is low and you can have protein precipitation.
Probably when you add the loading buffer the precipitate it will be re-solublized thanks to SDS but however, if you can avoid protein precipitation is certainly better.
ciao
Manuele
In electrophoresis, you want the concentration of extraneous salts to be as low as possible. Therefore, it is best to use water for dilution. The pH is fixed by the SDS-sample buffer.
Agreed with Engelbert Buxbaum , if you don't have lysis buffer left, just use water, it should work.
I wouldn't use lysis buffer, the ions interfere with electrophoresis. For small dilutions, you can get away with dd water, for stronger dilutions (say, more than 2-fold) I'd use 1 x SDS-sample buffer. Otherwise, the glycerol conc would become so low that sample loading into the well becomes more difficult.
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