Re: Cyclin D1

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From:Phyllis Davie <> (by way of histonet)
To:histonet <>
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Hoo-Boy!  Wouldn't I love to hear that information too.  I have just
placed an order for my 5th clone of Cyclin D1.  They all "work"
(sort-of), but none at a level that I would call good.  I'll give you a
run-down of what has worked for me, but I would sure like to hear from
others too.

clone P2D11F11 (Novocastra cat# NCL-CYCLINC1-GM) 1:250.
 Pretreat by steaming 20 minutes in 6mM citrate, pH 6.0, followed by a 20
minute cooldown.  Rinse in water, then digest 10 minutes in 0.1% pronase
(w/v in PBS) at room temperature.  Rinse again, then proceed.  This
treatment hereafter referred to as S20/+10.

clone DCS-6  (Dako cat# M7155)  1:100  S20/+10

clone 5D4   (Immunotech-Coulter cat# 1740) 1:1000  S20/+10

clone DCS-6 (same clone as DAKO, but from NeoMarkers, cat# MS-210-P0)
1:500, S20/+10

clone AM29   (Zymed cat# AS18-0220)  1:100,  Pretreatment in a microwave
pressure cooker in the citrate for 10 minutes at pressure, followed by
cooldown until 60 degrees C.

clone DR1BC2   (	BioCare Medical	CM090A or CM090D)  ON ORDER (the
recommends using their "EDTA Decloaker" antigen retrieval buffer for best

   I have tried a wide variety of other pretreatment options with varying
buffers, pH's, times, heat, no-heat, enzymatic digestions, ... anything I
could think of.  The most recent thing I heard was that a Tris/EDTA
buffer at pH 8.5 would do the trick, but I don't know concentrations of
the salts.  We gave it a try with 100mM Tris and 10mM EDTA for 20 minutes
in the steamer.  It gave results that were "working", but no better than
my S20/+10.  If anyone knows the magic antibody or pretreatment, I would
dearly love to hear about it.

>Anyone have any recommendations for good results with cyclin D1. Which
>company's antibodies and which method works best for you?
>Thanks in advance, J:>)

Phyllis Davie
Clinical Laboratory Supervisor
PhenoPath Laboratories
Seattle, WA
(206)374-9009  phone
(206)374-9009  fax

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