IEF of plant proteins

[naincy]

Hi,
Iam working on wheat total seed proteins. Presently Iam engaged in 2D electrophoresis of total seed protein. But Iam facing problem with IEF. Iam focussing IEF strip at 26,000 Vhrs.But I am not getting good gels. Kindly help
Regards, naincy

[kamil]

Give some more details about what is wrong with your gels. which pH gradient do you use, etc. It can be caused by lipids content. Do you precipitate the sample?

[naincy]

thank you very much for your reply.
Iam using pH range 3-10 but i am thinking to try pH range 4-7 as well.
Secondly can you please tell me if i can use pH 4-7 ampholyte alongwith pH 3-10 ampholyte while using IPG strip of 3-10 pH range.

bye nd tk cre
naincy

[kamil]

Hi, I can tell you, that IPG 4-7 is radically better than IPG 3-10 and you can see probably the same range of proteins on both (the focusing on IPG 3-7 ends on pH 7 approximately). About ampholytes, I do not know, if I get your point, but in IPG lysis buffer I use the same ampholytes for both IPG strips. The important thing is: do you precipitate your proteins before loading on the strips? IPG 3-10 usually requires sample precipitation, specially if your sample contains big amounts of lipids or some others contamination. IPG 4-7 does not require this procedure.

Good luck.
Kamil

[naincy]

Hi dear,
yes i do use same pH range ampholyte. Recently i read that if 4-7 pH range ampholyte is mixed with pH 3-10 ampholyte in the rehydration buffer then the resolution can improve.
Yes i do precipitate my plant sample before dissolution in lysis buffer.

Regards

Naincy