5 replies to this topic

[Myco Rax]

Hi All,
Can one use a colloidal coomassie stained  SDS-PAGE gel for the western blotting?
I mean, once the gel is coomassie stained, then can we destain it with destaining solution and use the same for the western blottin?
Have anyone tried it.
Please give your suggestions about it.
Thank you

[mdfenko]

the coomassie stain may interfere with the western, especially if you develop with color deposition.

the coomassie will, at least partially, come off the protein when transferred but will bind to the membrane.

[Myco Rax]

mdfenko, on 15 July 2011 - 08:38 AM, said:

the coomassie stain may interfere with the western, especially if you develop with color deposition.

the coomassie will, at least partially, come off the protein when transferred but will bind to the membrane.

Hi,
What if I completely destain it?
Thank you

[Prep!]

if u just wanna identify ur protein of interest then u can go ahead.. hoping that the antibody binding site is secure!!!

[mdfenko]

Myco Rax, on 17 July 2011 - 11:40 PM, said:

What if I completely destain it?

can you? i think that may be very difficult.

you can transfer the stained protein (it will destain some during transfer). it's just that it will interfere with seeing color deposited on the membrane. if you are looking for fluorescence or chemiluminescence then you should have no problem.

[knuf]

For me, the amount of protein necessary to see by coomassie is way higher than that needed to see by western blot.