assorted nonsense
Of sexual matters: First, call me a prude but I must confess. I have never had pillow talk with the lab manager unlike some bloggers who shall not be mentioned. Of course there is nothing more erotic than a whispered “overnight ligation” or “digested to completion” but I prefer to keep my professional and personal lives separate.
RTP Gossip: Which suave, sexy (formerly) Stratagene sales rep has not only changed jobs but is apparently ready to settled down with his sweetheart?
Lab related: Roche Protease Inhibitor Cocktail Tablets.
Do these actually work? I have no idea but I add them whenever I do a protein purification out of E. Coli or do a pulldown experiment. For all I know these are sugar pills but everyone knows about the placebo effect. If it makes you feel better who cares how it works? They come in two sizes “regular” and “mini”. I use the “minis” to save a little money. The smaller tablets dissolve a little bit faster giving you a complete cocktail of protease inhibitors. I use this product and I’m not really sure why. How is that for faint praise?
Publications: A rejected manuscript! The second go around of “submitted”, okay, “resubmitted” as I climb down the ladder of success. On a positive note, I’m still one step above the ridiculous “manuscript in preparation” that shows up on CVs.
SAB: What was wrong with the site last week?
Music: Gram Parsons Anthology
Book: ” Drive Like Hell” a novel by Dallas Hudgens (is “Hell” going to activate the obscentiy filter?)
October 13th, 2005 at 12:33 pm
It depends on what temperatures you work at. If you’re really anal about keeping all your samples at 4 degrees, a protease inhibitor is probably not of much value. However if your IP, ChIP, or enzyme activity assay requires steps at room temperature or above then they will slow degredation of your protein.
I never used them when I was purifying proteins routinely because the volumes I typically worked with were too large and I was doing drug studies with NMR and didn’t want to even think about how they may alter the interaction of drugs with my protein. Just keeping everything cold was sufficient . When I started doing ChIP assays they became more relevant because we would do many steps out of the cold room.
October 13th, 2005 at 1:30 pm
Cold? And doing NMR? Rii-ght.
But yeah, keep it cold during the prep, use omp-/lon- bugs, and if your purification protocol is good enough you shouldn’t have a problem. I always use excess PMSF when doing the bug lysis, but don’t add it after that. One ion exchange and one gel filtration is usually enough to purifiy away from everything, including the vast majority of proteases. Stuff that gets in after that is likely to be from bacterial contamination of the purified protein.
And I used to work on nuclear pore FG repeats which are notoriously susceptibe to proteolysis, so I think I know whereof I speak.
October 13th, 2005 at 3:28 pm
Well, we couldn’t keep it cold in the machine. But the purification took about 3 days, so in that time, there was a lot of room to heat up the protein and kill it.