Dear Steve,
You may also consider custom labeling of your primary antibodies by Molecular
Probes, Inc. or try our Protein Labeling Kits.
To obtain information on our kits, please visit:
http://www.probes.com/lit/feature/alexa/section4.html
For a protocol for antibody labeling using amine reactive dyes, see:
http://www.probes.com/media/pis/mp00143.pdf
To place an order for custom synthesis and to get pricing information, contact
us at [log in to unmask] .
Sincerely,
Nataliya
--
Nataliya Panchuk-Voloshina
Product Manager, Biochemistry
Molecular Probes, Inc.
(541) 465-8354
(541) 344-6504 (FAX)
[log in to unmask]
http://www.probes.com
Mario Moronne wrote:
> Steve,
>
> You may not want to try this approach given the small amounts of
> material you have, but it is very easy to biotinylate one of your
> primaries (assuming at least some purification) using a succinimidyl
> ester (Molecular Probes or Pierce). After labeling with the
> non-biotin primary and secondary, you can then use your
> biotin-primary with streptavidin-fluor or its cousins.
>
> In the same context, you could directly fluor label each primary with
> a different color and do the labeling in one step. Again to be
> practical, the antibodies need to be moderately purified. Using
> succinimidyl esters and small salt exchange columns (Pierce has some
> ready made) for removal of excess fluors, you can prepare the
> conjugated antibodies in a couple of hours. It's really not hard.
>
> Caveats: 1) if you get it wrong, you can kill the primary
> 2) if the primary isn't purified it may be hard to get the fluor on
> because of background competition
> 3) if you've only got 20 ug of Ab, then you get one shot.
>
> Potential Pluses: One blocking step, one primary incubation, one wash
> sequence, probably the lowest non-specific background
>
> I think that people should consider labeling their primaries more
> often. It may not be appropriate for you, but I think in many cases
> like yours it can simplify multiple labeling protocols with only
> modest effort.
>
> >I am doing some labeling of tissues with two rabbit derived antibodies,
> >one against serotonin and one against a peptide called Tpep. I need to do
> >a double labeling experiment in order to determine whether both these
> >antigens reside in the same or different neurons and their axons.
> >
> >Two questions
> >
> >1. Can anyone suggest a reliable method for double labeling using a
> >secondary fluor-conjugated probe with two different antibodies derived
> >from the same species (rabbit)? I don't have a large supply of either of
> >these antibodies.
> >
> >
> >Steve Kempf
> >
> >_________________________________________________________________________
> >The mind is like a parachute; it works much better when it's open.
> >~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> >
> >Stephen C. Kempf Tel: 334-844-3924
> >Associate Professor Fax: 334-844-4065
> >Depart. of Zoology and Wildlife Science
> >101 Cary Hall
> >Auburn University, AL 36849
> >USA Email: [log in to unmask]
> >Director - AU Hybridoma Facility - www.auburn.edu/research/hybridoma/
> >
> > __________ /|
> > / o \ /-|
> > / _ \/--|
> > \___/ | DARWIN /\--|
> > __/ / \-|
> > \_________/ \|
> > | |
> > \_| \_|
> >_________________________________________________________________________
> >~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>
> Mario M. Moronne, Ph.D.
> Material and Life Science Div. M/S 6-2100
> University of California
> Berkeley Lab
> 1 Cyclotron Rd.
> Berkeley, CA
> 94720
>
> ph (510) 486-4236
> FAX (510) 528-8076
> [log in to unmask] or [log in to unmask]
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