frezzing sample and Co-IP - ask a question (Jul/01/2008 )

ask a silly question here:
If I have to freeze the cell lysate at -80 due to emergency, can I still use it for Co-IP? and whether it will change the output?
Thanks for any help.

Although not ideal, you can try the IP from your frozen lysate. Be warned that this can create changes in protein binding and you may not get the correct result. If you do get a good result, you need to repeat it anyway so I'd make sure at least one of the repeats was from fresh lysate. If you don't get good results, it may be from the lysate having been frozen and you'll want to repeat with fresh lysate anyway. Seems like at least trying the IP won't hurt.

You are wanting to determine the protein concentration of all your samples and, although Bradford isn't perfect, it does give good relative concentrations of multiple samples so you would want to determine the protein concentration of all your samples at one time with the same standard curve. I was going to say that if it is a small time course you could make your lysates and keep them on ice until the final sample but if you need a 24hr, this makes it impossible. Freeze your lysates, thaw on ice, respin to remove precipitants and then bradford. Make absolutely certain you are using phosphatase inhibitors. I can't recommend AEBSF over PMSF enough. The half-life is twice as long. The only other thing I can suggest is for long time courses I just freeze the plate of cells at -80. I wash the cells 3X in PBS, remove as much PBS as possible and put the plate in the -80 freezer. The cells instantly freeze so there is no change in enzymes or phosphorylation. I then just harvest all the samples at once (on ice or in the cold room). I've kept these plates in the freezer for months and been able to harvest good western samples with no problems. It's also a good way to keep control cells on hand at all times.

Thanks for the help, I appreciate it. You briefly said AEBSF over PMSF and said the half life is longer. Is the half life of PMSF really short, I have no clue. And which phosphatase inhibitors would you recommend? My phospho antibodies aren't that great so I want to make sure the samples are perfect. Thanks again, really appreciate it.

The stability of PMSF (and AEBSF) greatly depends on the temperature of the solution. This is partially why some people believe you should only make lysates in the cold room but I hate standing in there for hours on end and I've never had any problems with a bucket of ice on the bench. As for the actual half-life, I've just always heard it's approximately twice as long but even the Sigma catalog notes that AEBSF is preferable to PMSF due to better water solubility and stability. I always add AEBSF (1mM), Complete (Roche) 1X, sodium vanadate, a tyrosine phophatase inhibitor, and sodium fluoride, a ser/thr phosphatase inhibitor. Although I've seen quite a wide range of concentrations in different protocols/recipes, personally I use them at 2mM and 10mM respectively. I've seen concentrations as high as 200mM and 50mM though.

Good luck with the phospho-antibodies. They can be tricky and even small changes can make huge differences. Some prefer PVDF over nitrocellulose whereas others are the opposite. Some prefer blocking in BSA, others milk. It may be worth your time to run a few gels and just try different conditions to see if one isn't better.