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

[arumugam]

Hi

As compare to other protein , plant protein are creating many problem in 2D gels. Let me know that how many ipg strips your running in single time, is it reaching proper voltage ( ramping) in all the steps, quantity of protein loading, and also rehydration of the strip ( passive or active) and sample loading ( cuploading method) may improve your separation.

[naincy]

Thanx for ur reply......but i guess now m gwttin good 2D gels....there is a bit of streakig but that is normal i think..
regards
naincy

[samratlogan]

hi nancy, im logan here.. i need a huge favour from u nancy.. it would be very grateful if u could help me in this issue..

me too doing my 2d in plant protein... mine is the rice plant (oryza sativa). When i run my protein samples in sds-page, i could obtain a very clear and good gel image resolution... The bands are clear.

But when i run it in 2d electrophoresis, i could not obtain any spots more than 10 spots.. only few spots are visible.. less than 10.. im using ph 4-7 strip, and ampholyte ph 3-10..

Please do advice me on this issue.. im trying to solve this for past 5 months but still couldnt.

thank you

[naincy]

hi dear,

I hope you are working on whole proteome??? or if any specific protein then please tell.

i guess u would be adding protease inhibitors like PMSF or inhibitor cocktail when you are extracting proteins. Kindly tell me which 2D system you are using biorad or amersham??? Secondly tell me the composition of your protein solubilization buffer. When you say that 1D SDS is normanl but you are not getting spots in 2-De that means that either your protein is degrading or there is a problem with your rehydration buffer.

Naincy

[samratlogan]

Thank you very very much for ur reply...

Yes, im working on whole plant proteome.. And im using biorad mini protean tetra cell... I dont think so the protein is degraded because if i run the same sample in SDS, it shows no degradation.. its fine... but only in 2 D , it gives problem....

I used protease inhibitor cocktail in all my extraction buffer. Im currently working on different plant protein extraction methods to choose the best extraction method... so my extraction buffer varies...

In one of my extraction buffer i hv used PMSF... Could u advice me how am i supposed to place the strip while running the second dimension... After IEF Focusing... The gel side of the strip should be facing the spacer plate or the thin plate???

Thank you very much in advance