5 replies to this topic

[jaknight]

I am trying to use dialysis to remove a low-molecular weight compound from cell lysate. However, about 90% of the total protein precipitates during dialysis.

I am dialysising using PBS at 4degrees.

I haven't done dialysis before so maybe I'm making a rookie mistake, but it seems very strange to me.

I can do dialysis with just about any buffer. The salt concentration of my sample isn't important, so if anyone has any suggestions I'd be very greatful.

I should also say that I'm not trying to denature the sample at all. I'd like to try and keep the proteins fully natured.

Edited by jaknight, 14 July 2009 - 12:16 PM.

[mdfenko]

you may be precipitating proteins that are not normally soluble (eg membrane proteins, cytoskeletal proteins) with pbs.

can you dialyze against your lysis buffer? or are you trying to remove those components?

Edited by mdfenko, 15 July 2009 - 07:51 AM.

[jaknight]

I could dialyze against my lysis buffer and see if that helps. My lysis buffer is just NP40 buffer. It's a drug that I am trying to remove from the sample, so the lysis buffer is okay for me.

If I am precipitating insoluble proteins, would these account for 90% of my sample? That's about how much I am losing. The cell lysate is from differentiated C2C12s.

[mdfenko]

jaknight, on Jul 15 2009, 12:32 PM, said:

If I am precipitating insoluble proteins, would these account for 90% of my sample? That's about how much I am losing. The cell lysate is from differentiated C2C12s.

possibly. i can't speak about a specific cell line but, in general, there is a lot of protein in the membrane and cytoskeleton.

[Wil]

What about doing the procedure at room temperature!
It is a well-known phenomenon in antibody dialysis that some do precipitate at 4°C and not at RT.

[Hong Zhan]

Wil, on 17 July 2009 - 06:21 AM, said:

What about doing the procedure at room temperature!
It is a well-known phenomenon in antibody dialysis that some do precipitate at 4°C and not at RT.

Is that true???? I have same issue... IgG (anti-myc). I want to remove glycerol from the buffer, and do conjugation... At RT and 4C I both saw the precipitation in dialysis buffer PBS.... I guss maybe it's the crystalization of the salt???...