Western blot trouble

Scott Brown via methods%40net.bio.net (by SBrown from ccia.unsw.edu.au)
Sat Jan 30 00:57:24 EST 2010


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: sbrown from 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.



More information about the Methods mailing list