3 replies to this topic

[jakatta70]

Hi,

Has anybody ever performed western blotting for NFkB? specifically on cells that were seeded into culture plates? I will be doing this on 150,000 Caco-2 cells quite soon. I have been told by a girl I work with that I wont be able to get enough of the protein and she advises using a T75 flask.

Now, the problem with that is that I have an offensively large numbers of treatment groups and I dont fancy wasting all my cells. So would I be able to get enough NFkB from this? The antibody will be recognising total protein and no phosphorylated form.

[Dr Teeth]

jakatta70, on Sep 1 2009, 10:39 AM, said:

Hi,

Has anybody ever performed western blotting for NFkB? specifically on cells that were seeded into culture plates? I will be doing this on 150,000 Caco-2 cells quite soon. I have been told by a girl I work with that I wont be able to get enough of the protein and she advises using a T75 flask.

Now, the problem with that is that I have an offensively large numbers of treatment groups and I dont fancy wasting all my cells. So would I be able to get enough NFkB from this? The antibody will be recognising total protein and no phosphorylated form.

You may not have enough protein to detect NfFkB with a total cell lysate from only 1.5x10^5 cells, but it depends on the amount of NfKB in those cells, the antibody/detection sensitivity, and your total protein concentration.  A few options:

1) try decreasing the total volume of your lysis buffer to maximize protein concentration
2) try using a more sensitive detection kit (Pierce's supersignal ECL femto kit can detect ng amounts of protein) or super signal secondary antibodies that amplify your primary signal more than usual
3) try crude purifications rather than total cell lysates (e.g. nuclear/cytosolic extracts, immunopurifications, etc.)

[jakatta70]

Dr Teeth, on Sep 1 2009, 04:07 PM, said:

jakatta70, on Sep 1 2009, 10:39 AM, said:

Hi,

Has anybody ever performed western blotting for NFkB? specifically on cells that were seeded into culture plates? I will be doing this on 150,000 Caco-2 cells quite soon. I have been told by a girl I work with that I wont be able to get enough of the protein and she advises using a T75 flask.

Now, the problem with that is that I have an offensively large numbers of treatment groups and I dont fancy wasting all my cells. So would I be able to get enough NFkB from this? The antibody will be recognising total protein and no phosphorylated form.

You may not have enough protein to detect NfFkB with a total cell lysate from only 1.5x10^5 cells, but it depends on the amount of NfKB in those cells, the antibody/detection sensitivity, and your total protein concentration.  A few options:

1) try decreasing the total volume of your lysis buffer to maximize protein concentration
2) try using a more sensitive detection kit (Pierce's supersignal ECL femto kit can detect ng amounts of protein) or super signal secondary antibodies that amplify your primary signal more than usual
3) try crude purifications rather than total cell lysates (e.g. nuclear/cytosolic extracts, immunopurifications, etc.)

Many thanks for that reply. It will undoutbedly be useful. I will try the options you have advised.

Edited by jakatta70, 02 September 2009 - 05:48 AM.

[amina_Amy27]

Hi ,
I want to perform western blotting of NFkB in rat skin . Can anyone guide me with the protocol for protein extaction from skin tissue . Also can i replace nonidet p40 with triton x 100 ? Please reply I'd be greatful