Western blot trouble

Pow Joshi via methods%40net.bio.net (by pow.joshi from gmail.com)
Mon Feb 1 13:54:32 EST 2010


On 30 January 2010 00:57, 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?
>

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: 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.
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>


More information about the Methods mailing list