Conjugating antibodies - Kits/tips? (Oct/03/2005 )

I'm looking to conjugate my currently unlabelled antibody with Alkaline phosphatase.

I've seen two kits which I think will allow me to do this (Pierce EZ link activated AP kit and Freezyme Conjugate Purification Kit), but we've never tried this in the lab before.

Has anyone used these kits and do they work? Or is there somthing better out there?

Thanks
Rosie

Hi Rosie,
Sorry I wasn't much help in identifying your ELISA problem. Pierce sell reagents for labelling proteins with biotin and HRP and alkaline phosphatase (http://www.piercenet.com/products/browse.cfm?fldID=10A91438-58CB-4F53-BC3F-87BFF600A001).

Best wishes,
Ceri

Hi Rosie,
I think that probably is the problem as Pesji said. The primary ab specific to PKC is bound to the plate and the secondary anti-rabbit/mouse ab binds the bound primary ab. The secondary abs are normally would be something like an biotinylated anti-PKC ab plus streptavidin linked to the enzyme. You could use a secondary against PKC from a different species from the primary ab then anti-that species conjugated with Alkaline phosphatase.

I've never labelled any abs but Pierce is a big company and I'd guess their kits would be good. Another post-doc who has about 20 years experience said he's labelled abs with NHS-FITC so I guess this is an amine labelling reaction. So if you can get hold of NHS-AP I could probably get you a protocol. At the end of the day having a kit gives you an optimised protocol and all the reagents and should allow you to get your money back or at least technical help if it doesn't work.

The Biocompare website gives you comparisons of kits and reagents if you want to see what other kits there are (http://www.biocompare.com/jump/165/Protein-Labeling.html). The kit from Pierce is £345 and the activated AP is £167. Maybe you could just use the activated AP and make up your own solutions from their instruction booklet.

I wonder though if it would be cheaper to buy another ab from a different species (say a mouse monoclonal against PKC) to coat the plate as a primary ab and keep your current rabbit polyclonal ab as the secondary ab with your goat anti-rabbit-alkaline phosphatase. There must be mouse monoclonal antibodies out there that work and they would probably be cheaper that the £200 to label your existing ab with alkaline phosphatase.

Hope that helps.

All the best,
Ceri

Hi Rosie you can go on with the Pierce labelling kit it's agood product a graduate student used it succesfully for labelling monoclonal with alkaline phosphatase !

Pesji