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Expressway to Discovery
G Cn po eiaee o
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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
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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.
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TF-DetectTM Human p53 Activity Assay Kit User Manual
0
1
2
3
0 10 20 30 40 50 60 70 80
OD
450
MCF-7 Nuclear Lysates (µg)
P53 activity in MCF-7 Cells
0
1
2
3
0 2 4 6 8 10 12 14 16 18 20
OD
450
HEK293 Nuclear Lysates (µg)
P53 activity in HEK293 Cells
Figure 3. The activity of p53 proteins from the nuclear extracts of MCF-7 (Top) and HEK293
(Bottom) cells were detected using the TF-Detect p53 Activity Assay Kit. Both cell types were
treated with 0.2mM H2O2 for 3 hours before harvesting. The cell nuclear extracts were prepared
following the Preparation of Nuclear Extract protocol in the Appendix.
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TF-DetectTM Human p53 Activity Assay Kit User Manual
Components
Quantity
Storage temperature
p53 antibody
15 µl
-20°C Stable for at least 6 months
HRP-conjugated IgG
15 µl
-20°C Stable for at least 6 months
Recombinant p53 protein* (0.25µg/µl)
20 µl
-20°C Stable for at least 6 months
Dithiothreitol (DTT) (1M)
100 µl
-20°C Stable for at least 6 months
10x Binding Buffer A
1.5 ml
-20°C Stable for at least 6 months
10x Binding Buffer B
1.5 ml X 2
-20°C Stable for at least 6 months
10x Wash Buffer
25 ml
4°C Stable for at least 6 months
TMB Substrate Solution
12 ml
4°C Stable for at least 6 months
Stop Solution
12 ml
4°C Stable for at least 6 months
96-well p53 assay plate (12 strips)
1 plate
4°C Stable for at least 6 months
Reagent
1 well
1x Wash buffer
2 ml
1x Binding buffer A (without DTT)*
50 µl
1x Binding buffer A (with DTT at 1:500 dilution)**
50 µl
Sample nuclear extract
10 µl
Recombinant p53 (positive control and protein standard)
10 µl
p53 antibody in 1x Binding buffer B (1:1000)
100 µl
HRP IgG antibody in 1x Binding buffer B (1:1000)
100 µl
TMB substrate
100 µl
Stop solution
100 µl
II. Kit Components and Storage
Cat. No. TAK-P53-196 (1 plate, 96 reactions)
*Recombinant p53 can be used as both a positive control and a protein standard.
Materials required but not provided
5 ml and 10 ml graduated pipettes, beakers, flasks, and cylinders
10 μl to 1,000 μl adjustable single channel micropipettes with disposable tips
50 μl to 300 μl adjustable multichannel micropipette, disposable tips, and reservoir
Micro-plate reader capable of reading at 450 nm (620 nm as optional reference wave length)
III. Preparation of reagents
Prepare a little bit extra than required amount to make sure enough buffers are available for
experiments. Required amount of reagents per well:
*Since DTT inhibits the tetramerization of p53 recombinant protein, 1x Binding Buffer A (without
DTT) is used for the dilution and binding reaction of the recombinant p53.
**1x Binding Buffer A (with DTT) is used for the dilution and binding reaction of sample nuclear
extracts.
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TF-DetectTM Human p53 Activity Assay Kit User Manual
Recombinant p53
solution
Binding Buffer A
(without DTT)
p53 standard
concentration
Final
volume
A
1 µl of p53 stock (250 ng/µl)
49 µl
5 ng/µl
50 µl
B
25 µl of A
25 µl
2.5 ng/ µl
50 µl
C
25 µl of B
25 µl
1.25 ng/µl
50 µl
D
25 µl of C
25 µl
0.625 ng/µl
50 µl
E
25 µl of D
25 µl
0.312 ng/µl
50 µl
F
25 µl of E
25 µl
0.156 ng/µl
50 µl
G
25 µl of F
25 µl
0.078 ng/µl
50 µl
H 0 60 µl
0 ng/ µl (blank)
60 µl
Wash buffer
Warm up 10x Wash Buffer to room temperature and mix well before use. If crystals have formed
in the 10x buffer, warm and mix gently until they have completely dissolved. To prepare 200 ml of
1x Wash Buffer for one 96-well plate assay, add 20 ml of the 10x Wash Buffer into 180 ml distilled
or deionized water. Mix gently to avoid foaming. The pH of the final solution should be adjusted to
7.8. The 1x Wash Buffer is stable for 30 days at 4°C.
Binding buffer A and B
Warm up 10x Binding Buffer A and B to room temperature and mix well before use. To prepare
100 ml of 1x Binding Buffer, add 10 ml of the 10x buffer to 90 ml distilled or deionized water. Mix
gently to avoid foaming. The 1x Binding Buffers are stable for 30 days at 4°C.
For the recombinant p53 as protein standard and positive control, 1x Binding Buffer A without
DTT should be used, since DTT inhibits the tetramerization of p53 recombinant protein.
For nuclear extract samples, 1x Binding Buffer A with DTT should be used. DTT should be added
to 1x Binding Buffer A at 1:500.
Samples
We recommend using 10 µl of nuclear extract at 0.2 - 5 µg/µl for each sample well. The total
protein amount is 2-50 µg per well. Note: The recommended amount is provided as guidance. A
broader range of value may be used.
Protein standard curve
For those who wish to quantify the amount of p53 in their samples, GeneCopoeia offers
recombinant p53 as a protein standard. Starting with a 250 ng/µl working stock of recombinant
p53, make serial dilution in a range of 5 ng/μl to 0 ng/μl using Binding Buffer A (without DTT).
Note: the recommended dilution range is provided as guidance, a broader range of values may
be used.
10 µl of each protein standard concentration will be used to generate the standard curve. The
range of protein amount for the standard curve is 0 ng to 50 ng.
Positive control
If quantitation is not necessary, 10 µl of the 2.5 ng/µl p53 protein standard can be used as the
positive control.
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TF-DetectTM Human p53 Activity Assay Kit User Manual
Primary p53 antibody
Calculate the amount of primary p53 antibody needed to perform the desired experiments and
make 1:1000 dilutions with 1x Binding Buffer B (100 μl/well).
Secondary HRP conjugated antibody
Calculate the amount of HRP conjugated antibody needed to perform the desired experiments
and make 1:1000 dilution with 1x Binding Buffer B (100 μl/well).
TMB substrate solution
Take appropriate volume of TMB Substrate (100 μl/well) and warm it up to room temperature 1
hour before use. Note: Avoid direct exposure of TMB reagents to intense light and oxidizing
agents during storage or incubation. The TMB Substrate Solution may develop a yellow tinge
over time. This does not affect the product performance. A blue color in the TMB Substrate
Solution, however, indicates that it has been contaminated and must be discarded. After use,
discard remaining TMB substrate solution.
Stop solution
Prior to use, take appropriate volume of Stop Solution (100 μl/well). After use, discard remaining
Stop Solution.
IV PROCEDURE
Determine the number of wells and strips needed. Store the unused strips at 4°C.
1. Mix all reagents thoroughly yet gently to avoid foaming before use.
2. Rinse the 96-well plate with approximately 200 μl Wash Buffer per well. Empty and tap the
plate on absorbent pad or paper towel to remove excess buffer. Be careful not to scratch the
surface of the 96-well plate.
Note: Use the plate immediately after washing or place upside down on a wet absorbent paper
for not longer than 15 minutes.
3. Sample wells: Add 50 µl of Binding Buffer A (with DTT) to the wells, and then add 10 µl
samples (2-50 µg of proteins). Mix well. Duplicated wells for each sample are recommended.
Standard curve wells: Prepare p53 protein standard according to the instruction in Preparation of
reagent. Add 10 μl of p53 standard in duplicate to the standard wells and mix with 50 µl of
Binding Buffer A (without DTT).
Positive control wells (if protein standard curve is not performed): Add 10 µl of 2.5 ng/µl p53
recombinant protein per well. Add 50 µl of Binding Buffer A (without DTT) and mix well.
Blank wells: Add 60 µl of Binding Buffer A (without DTT).
4. Cover the plate with a plate cover. Incubate at room temperature for 1 hour by gently rocking
the plate. Remove the plate cover and empty the wells.
Wash the plate by gently rocking it for one minute using 200 μl Wash Buffer per well. Repeat
three times.
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TF-DetectTM Human p53 Activity Assay Kit User Manual
5. Add 100 μl of diluted p53 primary antibody to each well, including the blank wells. Cover the
plate with a plate cover. Incubate at room temperature for 1 hour by gently rocking the plate.
Remove the plate cover and empty the wells.
Wash the plate by gently rocking it for one minute using 200 μl Wash Buffer per well. Repeat
three times.
6. Add 100 μl of diluted HRP conjugated antibody to each well, including the blank wells. Cover
the plate with a plate cover. Incubate at room temperature for 1 hour by gently rocking the plate.
Remove the plate cover and empty the wells.
Wash the plate by gently rocking it for one minute using 200 μl Wash Buffer per well. Repeat
three times.
7. Add 100 μl TMB Substrate Solution to each well (including the blank wells) and mix well.
Incubate the plate at room temperature for about 5-15 minutes.
8. Add 100 μl Stop Solution to each well (including the blank wells) and mix well. Read the plate
within 5 minutes using a microwell plate reader at 450nm.
VI Reference
1. Hollstein M., et al (1999) Mutat. Res. 431:199-209.
2. Renard P., et al (2001) Nucleic Acids Res. 29: (4) e21.
VII Appendix
Preparation of Nuclear Extract
1. Aspirate medium from a 10 cm plate and wash the cells with ice-cold PBS.
2. Add 1 ml of ice-cold PBS (per 10 cm plate, 4 - 8 x 106 cells). Scrape the cells into PBS and
then transfer them into a pre-chilled eppendorf tube.
3. Spin at 4ºC, 2,000 rpm for 5 minutes. Discard the supernatant.
4. Resuspend the cell pellet in 200 µl of Buffer 1 (per 10cm plate, scale up or down
proportionally for other size culture vessels). Incubate on ice for 15 minutes to allow cells to
swell.
5. Add 20 µl of Buffer 2 to the cells. Vortex for 10 seconds. Then incubate on ice for 5 minutes.
6. Spin at 4ºC, 14,000 rpm for 3 minutes.
7. Transfer the supernatant (cytoplasmic proteins) into a new eppendorf tube.
8. Add another 200 µl of Buffer 1 to the cell pellet and mix gently.
9. Spin at 4ºC, 14,000 rpm for 3 minutes.
10. Transfer and combine the supernatant in the cytoplasmic protein tube from step 7.
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TF-DetectTM Human p53 Activity Assay Kit User Manual
11. Resuspend the pellet with 200 µl of ice-cold Buffer 3. Vortex for 30 seconds. (Optional:
Sonicate for 2-3 seconds to break down the pellet.) Then rotate vigorously at 4ºC for 30
minutes.
12. Spin at 4ºC, 14,000 rpm for 10 minutes.
13. Transfer the supernatant (nuclear proteins) into a fresh, pre-chilled tube. Measure the protein
concentrations. Leave on ice if use immediately, or store aliquots at -80ºC until use.
Buffer 1: 25 mM HEPES, pH 7.4
10 mM KCl
1.5 mM MgCl2
1 mM DTT
100 mM PMSF
Buffer 2: 10% IGEPAL
Buffer 3: 25 mM HEPES, pH 7.4
420 mM NaCl
25% Glycerol
1.5 mM MgCl
0.2 mM EDTA
1 mM PMSF
VIII Limited Use License and Warranty
Limited Use License
Following terms and conditions apply to use of TF-Detect Human p53 Activity Assay Kit (the
Product). If the terms and conditions are not acceptable, the Product in its entirety must be
returned to GeneCopoeia within 5 calendar days. A limited End-User license is granted to the
purchaser of the Product. The Product shall be used by the purchaser for internal research
purposes only. The Product is expressly not designed, intended, or warranted for use in humans
or for therapeutic or diagnostic use. The Product must not be resold, repackaged or modified for
resale, or used to manufacture commercial products or deliver information obtained in service
without prior written consent from GeneCopoeia. This Product should be used in accordance with
the NIH guidelines developed for recombinant DNA and genetic research. Use of any part of the
Product constitutes acceptance of the above terms.
Limited Warranty
GeneCopoeia warrants that the Product meets the specifications described in the accompanying
Product Datasheet. If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet
these specifications, GeneCopoeia will replace the Product. In the event a replacement cannot be
provided, GeneCopoeia will provide the purchaser with a refund. This limited warranty shall not
extend to anyone other than the original purchaser of the Product. Notice of nonconforming
products must be made to GeneCopoeia within 30 days of receipt of the Product. GeneCopoeia’s
liability is expressly limited to replacement of Product or a refund limited to the actual purchase
price. GeneCopoeia’s liability does not extend to any damages arising from use or improper use
of the Product, or losses associated with the use of additional materials or reagents. This limited
warranty is the sole and exclusive warranty. GeneCopoeia does not provide any other warranties
of any kind, expressed or implied, including the merchantability or fitness of the Product for a
particular purpose.
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TF-DetectTM Human p53 Activity Assay Kit User Manual
GeneCopoeia Products are for Research Use Only Copyright © 2010 GeneCopoeia, Inc.
Trademarks: GeneCopoeia™, TF-Detect™ (GeneCopoeia Inc.) TAKP53-120910
GeneCopoeia is committed to providing our customers with high-quality products. If you should
have any questions or concerns about any GeneCopoeia products, please contact us at 301-762-
0888.
© 2010, GeneCopoeia, Inc.
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