Problems in runnging Native gel - Problems in runnging Native gel (Oct/28/2009 )

Hi everyone, my protein has a pI above 9 , and it is 150kDa. I know I should run an acid native gel, I have tried to run in acetate system for 4 hours at 30mA, and the gel percentage is 7.5%, but it didnt go down, and stayed at 2-3mm from the well, and I observe some precipiate inside the well as well, so I tried to a higher pH and the protein still didn't move to the negative pole. Do anyone know what is the problem behind this?

As I am studying protein oligomerization and I like to study in physiological condition, so I prefer running in a neutral pH around 6-7 , is there any protocol for native running at pH 6-7?

Thanks alot

somewhere in the archives is the formulation for neutral pH native page. if you can't find it then let me know and i will repost it.

thanks alot

do you think the BN-PAGE can be used in my case?

bn-page will modify the protein. it may lose activity. you can try it.

the coomassie in bn-page will impart a net negative charge to the protein, like sds does, but without denaturing it, unlike sds.

i realized that i may not have posted the neutral pH native page formulation on this forum so here it is:

solutions(all percentages are w/v):

30%:0.8% acrylamide:bisacrylamide (for the running gel)

10%:2.5% acrylamide:bisacrylamide (for the stacking gel)

10x running gel buffer:
6.0ml 1M hcl
add some dih2o
adjust pH to 7.9 by dropwise addition of 1M tris base
add 0.46ml temed
bring to 100ml with dih2o

10x stacking gel buffer:
6.0ml 1M hcl
add some dih2o
adjust pH to 6.2 by dropwise addition of 0.5M imidazole
add 0.46ml temed
bring to 100ml with dih2o

60% sucrose

10% ammonium persulfate

4.0mg riboflavin in 100ml dih2o

10x electrode buffer:
26.29gm glycine
add some dih2o
adjust pH to 7.3 by dropwise addition of 1M tris base
bring to 1000ml with dih2o

running the gel:
prepare running gel to a percentage appropriate for your separation needs or two solutions for a gradient (a good starting point, if you are not sure of the size or mobility of the protein of interest, is 7.5% or a 5-20% gradient). start polymerization with ammonium persulfate.

prepare stacking gel (i recommend 3%):
for 8ml 3% stacking gel (you can adjust for any volume that you require)
2.4ml 10% acrylamide
0.8ml 10x stacking gel buffer
2.67 ml 60% sucrose
dih2o to 7ml
start polymerization with 1ml riboflavin and photopolymerize with a fluorescent light.

electrodes are set to run (-) to (+)

add some bromphenol blue (5ul of 0.05% should be more than enough for a large sample) and sucrose or glycerol to make the sample dense enough to layer on top of the gel.

we run at 5mA, constant current, overnight on a slab gel much larger than a minigel, i guess you can try 100V until it finishes but watch out for overheating.

i'm sorry that i can't give you the reference for the gel formulation but i entered it about 30 years ago and, at that time, didn't think to write it down (how stupid was that?!).

good luck

Thanks alot for your formulation , i shall try it these two days,
by the way, I am trying to separate them by doing the blue native agarose gel