Western Blot - unspecific antibody binding - (Oct/10/2006 )

Hi all,

I have done a western blot using a primary antibody (anti-beta actin) 1:10000 in 5% blocking buffer and a secondary antibody (peroxidase conjugated) 1:10000 in 1% blocking buffer, each of them with 1h of incubation. After the addition of DAB, I saw many unspecific bands. However, I did another western blot using another primary antibody (anti-GSTP1) 1:5000 in 5% blocking buffer and the same secondary antibody (peroxidase conjugated) 1:10000 in 1% blocking buffer, again each of them with 1h of incubation. And I saw the same band patterns of the western that I have done with the anti-beta actin antibody!!!!
I don´t have idea what is happening?!?!?!?!
Please, what do you think I should do???

Thanks all,
Carol

P.S: I attached a Word file with my membranes (beta-actin and GSTP1 immunodetection).

-Carolss-

Is your second antibody against your host?

-aspergillie-

and what's happening if you incubate directly your secondary antibody on a fresh membrane?
it seems already well diluted, but maybe you need to optimize its concentration.
...
I read a post on the bioforum where they said that kidney extracts contain denaturation-resistant peroxydases. What's happening if you add substrate on your membrane (without any prior antibody incubation)

-Missele-

QUOTE (Carolss @ Oct 10 2006, 05:25 PM)

I don´t have idea what is happening?!?!?!?!

Please, what do you think I should do???

Thanks all,

Carol

P.S: I attached a Word file with my membranes (beta-actin and GSTP1 immunodetection).

preincubate your blots with 0.1 % H2O2 ( in hard cases use NaN3) before blocking

-The Bearer-

Kosmo, what does the peroxide or azide pre-incubation do? Just curious.

-WAstate-

Thanks for all replies

Aspergillie, my secondary antibody is against rabbit IgG, and my two primary antibodies (anti beta-actin and anti-GSTP1) are rabbit IgG.

Missele, I'll try to do this optimization in the concentration of the secondary antibody. Fresh membrane means after the transference?

Kosmo, I'm also curious what does the peroxide or azide pre-incubation do?

thanks
carol

-Carolss-

yes, I mean a brand new one after transfer, not one that has been stripped.

-Missele-

QUOTE (Missele @ Oct 11 2006, 01:27 AM)

I read a post on the bioforum where they said that kidney extracts contain denaturation-resistant peroxydases.

Missele has a very good point here!
For instance from our bone samples, we have to use a second antibody that is not conjugated with AP, because that is also present in the samples.
You can also try to use a second antibody that is conjugated with an other tag than HRP.

-aspergillie-

QUOTE (aspergillie @ Oct 11 2006, 12:31 PM)

QUOTE (Missele @ Oct 11 2006, 01:27 AM)

I read a post on the bioforum where they said that kidney extracts contain denaturation-resistant peroxydases.

So, first try the substrate, directly on your membrane.
If there is nothing appearing, try to optimize the secondary antibody.
If you see bands while using directly the substrate, you need to change your detection method, or to neutralize the peroxydase on your membrane by treating with azide.
I don't know the conditions, but you should be able to find the post where someone suggested that and ask him.

-Missele-

Thanks all!!!!!
You´re helping me a lot!!!!!
I´ll first try incubate with the substrate and see what happen.
If I need to preincubate my blots with 0.1 % H2O2. Do you have idea how long???

Thanks
Carol

-Carolss-