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ELISA HELP NEEDED Cant Get anything to Work (Allison)

ELISA HELP NEEDED Cant Get anything to Work (Allison) - Protein Forum

ELISA HELP NEEDED Cant Get anything to Work (Allison) - Protein Forum


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Old 03-27-2007, 07:32 PM
Dan Guire
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Default ELISA HELP NEEDED Cant Get anything to Work (Allison)



Hi Barbara,

The main point has already been made: Your anti rabbit alphatase conjugate
can only detect the rabbit antibody down on the Maxisorp plate. It doesn't
matter whether there is antigen for the capture antibody or not. No doubt a
negative control, leaving out only the mouse lysate, would show you that.

The series Allison outlined looks fine; the enzyme-conjugated anti mouse
shouldn't bind unless there is mouse antibody bound to antigen there. My
point is that if you already have biotinylated anti ENAC of one species, you
can capture any antigen with the non-biotinylated anti ENAC of the other
species, then detect with the biotin and a streptavidin-enzyme conjugate.
And a negative control is a must :-)

DanG

P.S. Do you have any affinity column experience by any chance? I could use
some help in that arena...



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1. Re: ELISA HELP NEEDED Cant Get anything to Work (Allison)


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Message: 1
Date: Mon, 26 Mar 2007 15:10:05 -0500
From: Allison <[Only registered users see links. ]>
Subject: [Protein-analysis] Re: ELISA HELP NEEDED Cant Get anything to
Work
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Barbara wrote:


If your first Ab is RABBIT anti mouse and your second Ab is goat
ANTI-RABBIT then all you will detect is the coating primary Ab.

A better series would be:

Rabbit anti-ENAC
blocking protein
lysate
Mouse anti-ENAC
Enzyme-conjugated goat anti-mouse IgG

Whatever antibody you decide to use it is important to keep track of the
animal it was made in as well as its specificity.

I have used the Nunc plates for ELISAs and I find that they work well. NAYY

HTH
Allison






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  #2  
Old 03-28-2007, 08:27 PM
Allison
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Default affinity columns

Dan Guire wrote:


I have done a bit....what is your question? If I can't help I am sure
someone else can.
Allison
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