Detection Of PCNA In Primary Tumor Cells By Immunoblotting

February 02, 2017

Biomedical Eng
The University of Texas at Austin
Professor

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Company:

Novus Biologicals

Product Name:

PCNA Antibody (Rabbit Polyclonal)

Catalog Number:

NBP1-32075

The aim of this study was to quantify PCNA protein expression in primary mammary tumor cells isolated from transgenic mouse mammary glands. PCNA served as a marker of proliferating tumor cells. This antibody performed well in freshly isolated tumor cells; it displayed low background and no nonspecific staining.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Primary mouse mammary tumor cells

Primary Incubation

1:200 in 1% w/v BSA, 1X TBS, 0.1% Tween-20 at 4 C overnight with gentle rocking

Blocking Agent

1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk for 1 hour at room temperature with gentle rocking

Secondary Incubation

Anti-rabbit HRP-conjugated secondary antibody (1:2000) in 1% w/v BSA, 1x TBS, 0.1% Tween-20 for 1 hour at room temperature with gently shaking

Tertiary Incubation

N/A

Detection

Pierce ECL Plus

Results Summary

Primary tumor cells were isolated from early and late stage tumorigenic C3(1) SV40 TAg transgenic mammary glands. Protein was isolated by M-PER extraction reagent (ThermoFisher) and 20 ug of protein per sample was subject to Western blot analysis. PCNA levels were detectable in cells isolated from early and late state mammary tissues.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

With this antibody, it was straightforward to achieve specific staining and low background.

The Bad

We experienced no drawbacks; this antibody worked well on the very first try and continued to function reliably for our studies.

The Bottom Line

This antibody produced excellent results for analysis of freshly isolated primary cells by immunoblotting.

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