p-ERK..

[Bouchra]

I want to detect phosphorylated ERK, pIKpB, pJNK, so I extracted protein from RAW cells and I loaded 25 ug/well, but I did not get any phosphorylayed protein (no bands) on the film, while with GAPDH, I got the right band. Do I have to load more proteins to see the phosphorylated proteins?

the concentration of protein I have is low, can I concentrate my protein solution and redo the western blot? I'm afraid if I concentrate the protein solution I may have too much salt which could interfer with protein migration on SDS-gel, what do you think?
Thanks

BE

[kiki06]

Hello Bouchra,

I used to lyse my whole well in SDS-PAGE Laemmeli loading buffer and load it all directly. You should be checking phospho-ERK and ERK levels but I would first blot for phospho-ERK and then strip and reprobe for ERK.

Make sure you use phosphatase inhibitors and do most of the incubations (with antibody) in the 4C (4 degree celsius) cold room

dont worry about the salt just load the whole amount.

All these should get you a big bright band...

[Bouchra]

Hi, it's me again

Since the max volume we can load in small SDS-gel is 50 ul, I'm thinking to speed-vac my protein solution to be able to load max amount of protein but I heard that could affect the migration of protein in SDS-gel? Did any one speed-vac his protein solution and used it for western blot? Thanks for sharing your experience

[admin]

Hello Bouchra,
i usually dont speed vac ever because that really removes all the water and concentrates the proteins (however it also increases the salt).

I would simply lyse in 45-50ul of SDS loading buffer (laemmeli) and load the entire amount.

cheers

[chinmoyee]

Hello Bouchra
Speed Vac works, I hav used it, but if you have less number of samples to load then can fuse 2-3 wells to load more sample or can use 1.5mm-2.0mm spacer as well.Make sure to add phosphatase inhibitors in all steps starting from lysis. & don't leave the sample at RT anytime.
Good Luck

[Pokoke]

Hi all, I have the same problem, I usually culture the cells in 6 well plate, should I change into smaller plate since my gel only has maximum loading volume of 15 ul . If loading the entire volume how about protein concentration? should it determined before loading? and does the Laemmli buffer will interfere Bradford protein assay.

Thank you for any advice

SiJohn