20 volumes of distilled water means that the dialysis bag or cassette is immersed in a container that holds 20 times the volume of the sample inside the dialysis bag or cassette. Personally, I would use a much larger volume ratio, such as 100 to 1000.

@Adam B Shapiro Fine. I was confused with it. Tell me one more thing, I want to concentrate my protein through dialysis. For this I have to dialyze for 48 hrs. After 48 hrs, what will be condition of sample?

I tried, I added the sample half of the bag and immersed and the bag got filled with the surrounding solution and two layers were formed. Now I am confused will the whole bag is the protein or just the sedimented one?

You will not be able to concentrate your sample by dialysis against distilled water. In fact, the sample will become diluted due to the higher osmolarity inside than outside, as you saw.

The separation of layers is the result of not stirring the dialysis sufficiently, so that the water that entered the bag did not mix into the sample. The proper method is to use a magnetic stir plate and as large a stir bar as can fit comfortably in the bottom of the beaker. Stir the bath as fast as possible without endangering the bag. The agitation is necessary to ensure adequate mixing.

The stability of the sample over time is dependent on the nature of the sample. It is advisable to do the procedure in the cold (4 degrees C, typically).

#Adam B Shapiro! Then what will u suggest I should use except dialysis. According to my protocol, it's mentioned that do dialysis against 20 vols of distilled water. What will be the sample buffer and dialysis buffer in this case? And I have to do it for 48 hrs. How can I do it on magnetic stirrer in this case?

The purpose of dialysis is to equilibrate the low-molecular weight solutes inside and outside the membrane, while retaining the high-molecular weight solutes inside the membrane.

The reason for dialyzing against distilled water is probably to remove most of the low-molecular-weight substances, such as salts, leaving only the high-molecular-weight whey proteins. The proteins can be concentrated after dialysis by freeze-drying, spray-drying, ultrafiltration, or precipitation with acid or organic solvent.

In this method, there is no sample buffer, since the point is to remove all the low-molecular-weight solutes. However, if you want to have the dialyzed protein solution contain some other solutes, you can either add them from concentrated stock solutions after the dialysis is finished, or have the dialysis bath consist of the desired solution instead of distilled water.

I already described how to use a magnetic stirrer to do the dialysis step. Since it is desirable to do the dialysis at a cold temperature, you can do it in a cold room, if your facility has one, or in a refrigerator that is equipped with an internal electrical socket for plugging in equipment, such as the type with glass doors that is used for chromatography equipment.

@Adam B Shapiro, If u don't mind can I get ur email or any other contact link please.

One more thing, i want to ask is that can I reuse the dialysis membrane or it's made for just one time use?

One time use is highly preferable.