On 30 January 2010 00:57, Scott Brown <sbr...@ccia.unsw.edu.au> wrote:
> Hi all, > > I've purchased a santa cruz Ab which is usually not such a great thing in > my experience, i optimized it using a wt cell line and a cell line that over > expressed my protein of interest. The blot looked great. However, when i > came to use it on some xenograft samples i have non specific binding > everywhere and in between where each lane is and including the lane i have > grey/black smears all over the membrane. > Usually i dilute my primary with 0.5%milk/TBST, i increased the amount of > primary and diluted with 5%milk/TBST to try and get rid of some of this > background and bring up my bands a bit more but i got the same result, a > blot with grey smearing all over it. > > Does anyone have any trouble shooting tips, i'm not that new to westerns > i've never had this problem before but i cant think what else to do. > > Also the actin blot is clear and clean, so it has to be something to do > with the primary i'm thinking. > > Any ideas? > Well, a few things: 1. what is the lysis procedure you use, which protease inhibitors, and how stable is you protein of interest after lysis?; I'm guessing you would'nt have an accurate idea (as is the case with most proteins in a tissue sample).If you have bands appearing that are lower than the protein of your interest, then there's a good chance that the protein degrades during your lysis procedure. Ergo, I could suggest increasing the protein inhibitors in your tissue lysis buffer (provided you are'nt lysing your cells using the SDS-PAGE buffer). if high mol weight bands are present, then perhaps you would want to increase the amount or concentration or add another detergent to your lysis buffer; for this could be due to protein aggregation due to high concentrations? Thirdly, I would laos check the state of your xenograft: what sort of tissue you're using, whether there is already some sort of cell death present.... Hope that helps Pow > > Scott > > Scott Brown > PhD Candidate > Molecular Carcinogenesis Program > Children's Cancer Institute of Australia for Medical Research > High Street (PO Box 81) > RANDWICK NSW 2031 > AUSTRALIA > > Phone: +61 2 9382 1829 > Fax: +61 2 9382 1850 > Email: sbr...@ccia.unsw.edu.au > Web: www.ccia.org.au > Children's Cancer Institute Australia is the only independent medical > research institute in Australia solely devoted to research into the causes, > prevention and cure of childhood cancer. Our vision is to save the lives of > all children with cancer and eliminate their suffering. > The information contained in this message and any annexure is confidential > and intended only for the named recipient(s). If you have received this > message in error, please contact the sender immediately and destroy the > original message. > > > Children's Cancer Institute Australia for Medical Research is the only > independent medical research institute in Australia devoted to research into > the causes, prevention, better treatment and ultimately a cure of childhood > cancer. Our vision is to save the lives of all children with cancer and to > eliminate their suffering. > > The information contained in this message and any annexure is confidential > and intended only for the named recipient(s). If you have received this > message in error please contact the sender immediately and destroy the > original message. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
