CO- IP for nuclear protein - (Aug/19/2014 )
I am trying to isolate the nuclear protein from mesenchymal cell using the nuclear isolation kit from Active mottif and then using this for Co-IP. I have tried many times, but Iam not able to get the protein of interest. All I see is the IG bands on the gel. First Iam not sure if the nuclear proteins are being isolated. Can anyone help me in confiming this. If someone is doing the same procedure for mammanlian cells can you please help me out sorting this. Is this kit really good to isolate nuclear protein. Any suggestion would be helpful.
Ip should be possible, but you need to supply a lot more detail on protocols before we can help you trouble shoot the problems you are having. Details of antibodies will be helpful too.
Can you IP the bait protein effectively? If you can't do this then the co-IP won't work.
A good nuclear marker is nucleolin. You should determine if you have clean nuclear extraction by examining a cytoplasmic marker as well to ensure that you don't have cytoplasm in your extraction.
Thanks for the mail. The nuclear extract is from ch10T1/2 cells. According to the protocol, I first wash the cells with PBS with protease inhibitor, add the hypotonic buffer, wait for 15 min, add 25ul of detergent and spin it for 30s at 3000 rpm. discard the supernatent and to the pellet add 50 ul of lysis buffer, centrifuge at 14000 rpm for 3 min. This is how the protocol works from active active motiff company. Then I use the supenatent to do the IP using agarose beads as per the sigma protocol. I use the antibody for DMP1 to pull down the protein. In the end I dont see the protein at all.
My concern is will the nucleus get pelleted when I spin briefly for 30 sec as the protocol says. If not how long do I have to centrifuge to get the nucleus down. Please suggest.
You say rpm - does the protocol say rpm or does it say RCF (or g): rpm measurements are useless unless you also know the rotor diameter, so it could be that your 3000 rpm is pelleting the nucleus. However, you should be able to check this - take a small amount of your cells and repeate the process, but resuspend the pellet in PBS rather than lysis buffer, then examine it under a microscope - you should be able to see nuclei fairly well at 400x.
I would say - work on getting your IP to work before you start the nuclear fractionation. Check that your antibody will work (look at the datasheet for validation for IP), check that it is working for straight western detection too - if you can't see it on a western, then you definitely can't tell if your IP is working!
Thank you. I will try it out.