On Sat, 30 Jan 2010 16:57:24 +1100, "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? > >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=20 Hi! I think the problem appeared once you increased the antibody concentration. In our lab we use a minimum antibody concentration in 4% (w/v) milk/TBS-T. Usually a too high antibody concentration increases the unspecific background, so one should optimize the antibody concentration downwards. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
