protein-protein interaction-plz help - (Jun/06/2007 )

Morning Hasina. Sorry, I forgot to mention that I always add protease and phosphatase inhibitors fresh to my lysis buffer. My boss claims that this is just a waste of money but everyone in the lab uses them...we just don't tell him. As for my cell lysates, I wash the plate of cells three times with PBS making sure to get as much off as possible the last time and then I put the lysis buffer directly onto the plate. I swirl the plate around to coat all the cells and let this sit in the cold room for at least 20 mins. I then scrape all the gunk to an edge and transfer to an eppy. I pipette up and down about 12-15 times to help sheer the DNA and then centrifuge at 14K rpm/4 degrees/minimally 20 mins. I then transfer the supernatent to a clean eppy. There have been a few times when a large pellet indicates I didn't get good lysis (not enough buffer) so I'll resuspend the pellet in a bit of fresh lysis buffer, let it sit and recentrifuge. This is when I know I'm going to need as much protein as possible. If I have tons of cells and know I can spare a bit, I'll wash the cells and on the last wash scrape the cells off the plates. I transfer the PBS/cells to a conical and spin at about 2000rpm for 5mins. Carefully remove the PBS without disturbing the cell pellet and resuspend cells in lysis buffer. Let this sit on ice for 20 mins, transfer to an eppy and centrifuge as above. Only problem with this method is I have lost the pellet once or twice to the vacuum aspirator...soon followed by lots of loud cursing. I now use my pippetor when it gets close to the bottom.

As for your lysis buffer, don't drop the salt anymore! Physiological levels are around 150mM and many interactions require salt bridges. I use a low salt/no salt wash only when I'm trying to get rid of background but I've seen the interaction. I would also like to recommend a book called Using Antibodies by Harlow and Lane. One of the first things my boss made me do was read this book and it has tons of great tips and troubleshooting ideas for all assays using antibodies. Hopefully someone around you has a copy.

Otherwise, you might want to try reversing the IP. Pull with the HA tag and look for the GFP tag. Again, for some unknown reason, some interactions can only be shown in one direction. I can show a specific interaction by pulling with HA and looking for the myc tag but when I pull with myc, the HA isn't there. I know this interaction is for real because I've actually got three independent assays (2-hybrid, GST pulldown and endogenous protein IP) but now I'm trying to work with mutants and truncations. I have no idea why the IP only works in one direction but I've had a number of people say they've seen this before.

I also agree with just about everything rkay447 said. Also, if you are "searching for the right buffer", start a low stringency (like NP-40 buffer @ 150mM salt) and then slowly work up to something like RIPA buffer, which typically is much more stringent. Also, good antibody selection is the #1 most important step IMHO; and remember that antibodies that work in a Western will not necessarily work well in an IP and vice versa. Are you antibodies raised in-house? If not and you are using commercial antibodies, try another one for the same epitopes (and from another company too). I usually stick with antibodies from Santa Cruz, Abcam, or Upstate - there's usually something between the three of them that will work.

Not much to add here, but I hope that helps.

(also, shouldn't this topic be in another forum?)