I did IP (Immunoprecipitation) and all steps of western blotting. After the result i try to use Ponceau red to detect proteins but evertime when use it for IPed proteins on membrane it didn't work. And yes, ponceau is fresh and also i tried it for another membrane which contains total protein. So i have 2 questions: Why it's not working? And how can i detect the proteins?
Hi, I would check the gel for protein retention. You can stain the gel with Coomasie blue. Maybe your proteins didn´t transfer at all.
Hi Okan Akar,
One very important thing that you need to remember while using PVDF membrane is that you need to pre-activate the membrane by wetting it in 100% Methanol and then equilibrate it with Transfer Buffer. Once this is done, use this activated membrane to setup transfer of proteins from SDS-PAGE gel.
This is not required for Nitrocellulose Membrane. Without the activation, PVDF will never uptake any proteins.
Hope this helps and you see your IP Band :)
Good Luck!
Utkarsh
Sorry if I misunderstand, but by all steps of western blotting, do you mean you go through blotting with primary and secondary antibody, adding developing reagent, and exposing to film? I'm not sure if you can do Ponceau at that point because the membrane would be saturated with the blocking reagent. If you Ponceau stain before blocking and don't see any bands, then Coomasie staining the gel is an excellent control (you should definitely see bands for the antibody used for IP on either the gel or the membrane).
Sorry i think i didn't clear. İ took a result already. I just wanted to see bant profiles. Because there has to be a 6 bants but i have 5 bants. So with ponceau red i'm gonna understand when my friend cutting the membrane did he cut my result or something else. Utkarsh i activated the membrane ofcourse and like i said i have a result. Micheal, I do IP a few times and from all my membranes i took a result. But everytime when i ponceau staining i cannot see the bants. Yes after doing blocking, immunoblotting and ecl solutions and chemidoc, i use ponceau but we do that all the time. I mean we take a result or not after chemidoc, we stain our membranes with ponceau and we see the proteins. İ don't understand why i cannot see with my IPed proteins. Thank you for your time Caroline, Utkarsh and Michael.
If you don't see any band profile after Ponceau staining in your IP lane, this indicates that after the IP , a very small amount of protein has been pulled down which you cannot see unless you probe with a specific antibody and look for it.
The sensitivity of Ponceau stain is in the range of 100-1000ng. This means you need a minimum of 100ng protein in your IP lane to see the bands. Since you do not, I presume your IP has yielded lesser. But, if you IP has worked (even though inefficient), you may be able to still pick it up by Western as it has better sensitivity and can detect very small amounts (almost 10ng)
Thank you Utkarsh. That was explain so much things.Actually i started to IP with 500ug total protein. But i'm not sure after the precipitation how much ug protein left. Cuz i add 50 ul loading dye to beads. If i'm correct there should be still 500ug right?
You start with 500ug total protein. Even if your protein of interest is a highly expressed one, maximum efficiency of IP is about 30-40% (from my experience and others). It is wishful thinking to expect 500ug in the IP.
Besides, if 500ug is total protein, logically speaking, how will you ever recover 500ug of your protein in IP? It's impossible.
I thought as concentration but yes you're right. Talking with you about troubleshooting was too instructive for me. Thank you very much.
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