Re: Vector ABC Elite Kit and biotinylated primary antibodies

<< Previous Message | Next Message >> (Hermina Borgerink)
To:Gayle Callis <>
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Hi Gayle,

I have used the ABC Elite kit, although not extensively.  I prefer to
use the BioGenex Supersensitive kit because it uses Streptavidin rather
than Avidin with the enzyme label.  Streptavidin does not contain any
carbohydrates that are able to bind non-specifically to substances such
as lectin that are present in many normal tissues. It also has an
isoelectric point that is close to neutral as opposed to avidin's which
is 10 (and this means that avidin conjugates can produce non-specific
binding because they are positively charged under physiological
conditions). And if I remember correctly, I think Streptavidin has a
higher affinity for biotin than does avidin. Last but not least, I lke
to use the BioGenex kit because I do not have to remember to make up the
ABC  complex ahead of time.  Again, there is nothing wrong with the
Vector kit, I just prefer the BioGenex. I use the Streptavidin-Alkaline
Phosphatase kit rather than the Horseradish Peroxidase to avoid using
DAB to visualize my proteins.  I use the Vector Red kit because it
produces a bright red color that contrasts beautifully with the Mayer's
nuclear stain.  And that I make up myself.  I have as yet to find a
commercial Mayer's that works as well as "home made".


Gayle Callis wrote:
> After years of being a purist and non kit user, we have decided to branch
> out and work with a universal kit.
> The majority of our primaries (murine CD markers) are biotinylated, and
> would like to know any pros and cons on using the ABC Elite kit with these.
>  Loss of sensitivity, etc.
> We have found the kit to be excellent, particularly with nonbiotinylated
> antibodies, but also have a major stock of the others, and happen to like
> using them overall.  We have always diluted our own Strepavidin-HRP
> substrate, and found this kit to give cleaner results with non-biotin
> conjugated primaries.
> My thoughts are if the primary is biotinylated, it should make little
> difference, since secondaries are biotinylated also.  As long a dilution
> panel is performed with appropriate fixation and blocking, all should work
> well.
> What are your experiences?
> Gayle Callis
> Veterinary Molecular Biology
> Montana State University
> Bozeman MT 59717-3610
> 406 994-4705
> 406 994-4303

Hermina Borgerink BA, HT(ASCP)IHQ
Department of Pathology
Wake Forest University School of Medicine
Winston-Salem, N.C. 27157
PH (336) 716-1538
Fax (336) 716-1515

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