IEF of plant proteins

[arumugam]

The IPG strip plastic side will face in spacer plate the gel side will be in thin plate, so that u can get continuation with PAGE in 2nd dimension. The low number of spot may be due to low concentration of protein load. How much concentration your loading in IPG strip, in what method your eatimating the protein?

[naincy]

hi

If u are still in the process of standardization of a protocol then i would suggest you to try urea buffer for protein extraction. The details about ths method are given in Hurkman and Tanaka,2007. The article is about wheat endosperm protein extraction. this article helped me a lot.

U can use PMSF, its fine. And gel side of IPG strip should be facing thin plate.

All the best

I hope this helps

naincy

[samratlogan]

Hi arumugam, thank you for the information... Yes , all these while i thought i was placing the strip upside down.. but i was correct... but still im not obtaining any spots... i have only identified few spots till todate... but im going to try the hurkman and tanaka procedure as nancy said tomoro... Again thank you very much

[samratlogan]

Hi naincy, i will try the extraction method that you said tomoro and i will ask your kind help if there is a problem... Thank you very much naincy...

[samratlogan]

hi arumugam,

thank you very much for your information. I have already obtained spots in my gels and i think its good enough. U and nancy have helped me a lot.. Thank you very much again.. take care

[bhuaks29]

Hi !
I need a favour from u. I am working on whole leaf proteome of chickpea plant (leguminous crop). But the no. of spots are around 50-60 in 4-10 ph range in 7cm gel. I think the number is too less. how to increase the spot number?? please help...

[arnab_roy]

samratlogan, Please provide detailed of your sample preparation and 2DE protocol. Please upload the gel images also. It will help people to understand your problem better.