cell lysis and DNA - sticky (Aug/03/2007 )

HI
when I use RIPabuffer to do protein axtractin on my 293T cells most of the time I got a lot of junk DNA.
this very difficult to get ride of it even after centrifugation
SO I sonicat my lysate and speen it but I still got proble when I load my sample be cause I got smily band and the western are not pretty.
so I wanted to know how do you proceed once you add your lysate buffer on you cell pelle.
do you have a details procedure.
as, do you vortex? do you speen and take the surpernatant and sonicate and another speen or do you add the ripa buffer sonicate, speen, take the supernatant and add leamni buffer and sonicat then speen before loading?

thanks

It sounds like you are making your lysate correctly but how long do you sonicate and spin the sample? I have made lysates with RIPA buffer and never had an issue with DNA. I resuspend a cell pellet in at least 2X more buffer than cells (ie: 50ul cell pellet = 100 ul buffer) and let it sit for 20mins in the cold room. I then sonicate (if just doing a western) or pipet up and down about 12 times if doing a Co-IP or functional assay (ie: kinase assay). My sonication is with a probe sonicator for 3-10second pulses with 15 seconds between each pulse and the whole time on ice. I then spin my lysate at 14K rpm for at least 15-20 minutes in a cooling centrifuge. I always have a large glassy/white pellet at the bottom and a nice clear lysate. The problems you are having with a smiley band and your westerns are most likely not related to the lysate itself. When you load your gel, is the sample sticky, thick and difficult to load? Then you have DNA. If it's easy to load, you're fine. I've found that a smiley gel usually means it's being run too quickly. Back off the voltage and it'll straighten out. As for your westerns, there are many potential areas for problems. Western blotting can be a very difficult assay and it takes time and experience to get consistantly good results. Be sure to ponceau stain your membrane after transfer to make sure the gel ran well and fully transfered. If the membrane looks ok then you need to work on optimizing the western (blocking, probing, washing..etc.). Remember when it comes to western blotting, even small things can make a world of difference. Some antibodies prefer nitrocellulose over PVDF or vis versa, some antibodies only work well with TBST and not PBST. The point is that if you are able to load your gels easily and the membrane shows good seperation your problem is most likely further down the process rather than your lysates.

we sonicate the extracts and it makes them less viscous. Then we centrifuge them and collect the supernatant. You might have to sonicate a bit more till they are less viscous.