Re: [Histonet] SV40 HRP protocol HELP

From:James L Burchette

Dear Anne:

I use the SV40 (catalog # DP02) from Oncogene, a division of Calbiochem. A 
dilution of 1:500 consistently works well from lot to lot. 

For manual or autostainer platforms, pretreatment is with Dako's Target 
Retrieval pH 6.1 solution for 20 minutes in a 100 C water bath. Following 
a one hour primary incubation, the bound antibody is detected with HRP 
labeled Dako Envision Plus (30 minute incubation). I like using an 
enhanced DAB, such as Dako's DAB+.

I find this SV40 also works well on the Leica Bond Max slide stainer. The 
same 1:500 dilution is used and the Bond pretreatment is programmed for 20 
minutes with HIER solution #1 (aka ER1). Other than the pretreatment 
programming, their standard incubation schedule for all of the subsequent 
steps works well.

Best Regards

Jim Burchette

Catalog # DP02
Host: Mouse
Isotype: IgG2a
Immunogen: purified SV40 large T-antigen
Epitope: Within amino acids 83-128
Form: Liquid
Formulation: In 50 mM sodium phosphate buffer, 0.2% gelatin, pH 7.5.
Preservative: 0.1% sodium azide
Positive Control: SV80 or SV-T2 cells
Negative Control: 3T3 cells
Comments: Recognizes ~94 kDa SV40 large T antigen in SV80 and SV-T2 cells. 

Ref.: DeCaprio, J.A., et al. 1988. Cell 54, 275. Whyte, P., et al. 1988. 
Nature 334, 124. Mitchell, P.J., et al. 1987. Cell 50, 847. Dixon, R.A.F. 
and Nathans, D., 1985. J. Virol. 53, 1001. Simanis, V. and Lane, D.P. 
1985. Virol. 144, 88. Mann, R.S. and Carroll, R.B. 1984. Virology 138, 
379. Sarnow, P., et al. 1982. Cell 28, 387. Crawford, L.V., et al. 1981. 
Proc. Natl. Acad. Sci. USA 78, 41. Lane, D.P. and Crawford, L.V. 1979. 
Nature 278, 261. Carroll, R.B., et al. 1974. Proc. Natl. Acad. Sci. USA 
71, 3754. Tooze, J. 1973. Cold Spring Harbor, New York. Tegtmeyer, P. 
1972. J. Virol. 10, 591.

"Anne van Binsbergen"  
Sent by:
10/09/2008 08:05 AM

histonet ,

[Histonet] SV40 HRP protocol HELP

Hello Histonetters
please could someone out there (anyone) assist with a working protocol for
Immuno SV40 (HRP)
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