GeneCopoeia TAK-P53-196 User Guide
G Cn po eiaee o
TM
Expressway to Discovery
G Cn po eiaee o
TM
Expressway to Discovery
TF-DetectTM Human p53 Activity Assay Kit
For rapid and sensitive detection and quantification of active p53
Cat. No. TAK-P53-196 (1 plate, 96 reactions)
User Manual
GeneCopoeia, Inc.
9620 Medical Center Drive, #101
Rockville, MD 20850
USA
301-762-0888
866-360-9531
inquiry@genecopoeia.com
www.genecopoeia.com
© 2010 GeneCopoeia, Inc.
TF-DetectTM Human p53 Activity Assay Kit User Manual
USER MANUAL
TF-DetectTM Human p53 Activity Assay Kit
I. Introduction and Principle
II. Kit Components and Storage
III. Preparation
IV. Procedure
V. Limited Use License and Warranty
I. Introduction and Principle
When responding to diverse cellular stresses, tumor suppressor p53 regulates target genes that
induce cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. p53
protein binds to a consensus p53-binding site and activates expression of downstream genes that
inhibit growth and/or invasion. Mutations in p53 frequently occur in numerous types of human
cancers. The p53 mutants lose the tumor suppressor activity1 by failing to bind to the consensus
DNA binding site.
TF-DetectTM Human p53 Activity Assay kit enables fast and sensitive detection and quantification
of p53 in a 96-well format2. Double-stranded oligonucleotides containing a p53 consensus binding
site are immobilized in a 96-well plate. The p53 proteins present in nuclear extracts are captured
by the immobilized oligonucleotides specifically and then detected by a p53 antibody and a HRPconjugated secondary antibody. The colorimetric signal generated by HRP substrate TMB can be
easily quantified by spectrophotometry.
A purified recombinant human p53 protein is also provided in the kit for use as protein standard
for quantifying and comparing p53 activities of different sample types or time points.
Sensitive – Detects as little as 0.8 ng of human p53 protein per well and performs better
than a similar competitor kit (Fig.1).
HTS compatible - Optimized for use on 96-well plate readers for high-throughput
screening assays. Single strip (8-well) assay can also be performed.
Fast - 3.5 hours from preparation to detection.
Quantitative – The active and purified human recombinant p53 protein provided in the kit
allows users to generate standard curve (Fig.2) and quantify p53 in samples.
Protocol Overview
Rinse 96-well plate (coated with p53 binding oligonucleotides)
Bind p53 (in nuclear extract or protein standard) to the immobilized oligonucleotides
1 hour
Wash 3X. Add p53 antibody
1 hour
Wash 3X. Add HRP-conjugated IgG antibody
1 hour
Wash 3X. Colorimetric reaction
------------------------------------Total ~ 3.5 hours
2
TF-DetectTM Human p53 Activity Assay Kit User Manual
0
0.5
1
1.5
2
2.5
3
0
0.78
1.56
3.13
6.25
12.52550
OD
450
P53 proteins (ng)
Comparison of P53 Activity Assay Kits
GeneCopoeia
Competitor
R² = 0.9839
0
0.5
1
1.5
2
2.5
3
0 10 20 30 40 50 60
OD
450
P53 proteins (ng)
Standard Curve of P53 Activity
Figure 1. Performance comparison between GeneCopoeia’s TF-Detect Human p53 Acitivity
Assay kit and a similar competitor product. A human recombinant p53 protein was detected and
quantified using both kits.
Figure 2. Standard curve for p53 quantitation is generated using GeneCopoeia’s purified p53
recombinant protein (included in the kit). The curve is provided for demonstration only.
3
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