Western blot trouble

watnne from googlemail.com via methods%40net.bio.net (by watnne from googlemail.com)
Sat Jan 30 12:44:08 EST 2010


On Sat, 30 Jan 2010 16:57:24 +1100, "Scott Brown"
<SBrown from 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: sbrown from 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. 


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