• Preparation of Lentiviral expression construct DNA:
Contents
1. 4 10 mM KCI 1 5 mM MgClo 1 mM DTT 10 mM PMSF Buffer 2 10 IGEPAL VII TF Detect Human p53 Activity Assay Kit User Manual Buffer 3 25 mM HEPES pH 7 4 420 mM NaCl 25 Glycerol 1 5 mM MgCl 0 2 mM EDTA 1 mM PMSF 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 repla2. M et al 1999 Mutat Res 431 199 209 Renard P et al 2001 Nucleic Acids Res 29 4 e21 Appendix Preparation of Nuclear Extract Aspirate medium from a 10 cm plate and wash the cells with ice cold PBS Add 1 ml of ice cold PBS per 10 cm plate 4 8 x 10 cells Scrape the cells into PBS and then transfer them into a pre chilled eppendorf tube Spin at 4 C 2 000 rpm for 5 minutes Discard the supernatant Resuspend the cell pellet in 200 ul 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 Add 20 ul of Buffer 2 to the cells Vortex for 10 seconds Then incubate on ice for 5 minutes Spin at 4 C 14 000 rpm for 3 minutes Transfer the supernatant cytoplasmic proteins into a new eppendorf tube Add another 200 ul of Buffer 1 to the cell pellet and mix gently Spin at 4 C 14 000 rpm for 3 minutes Transfer and combine the supernatant in the cytoplasmic protein tube from step 7 Resuspend the pellet with 200 ul of ice cold Buffer 3 Vortex for 30 seconds Optional Sonicate for 2 3 seconds to break down the pellet Then rotate vigorously at 4 C for 30 minutes Spin at 4 C 14 000 rpm for 10 minutes 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 80 C until use Buffer 1 25 mM HEPES pH 7
3. 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 It can be stored at 20 C for longer time 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 ul well TF Detect Human p53 Activity Assay Kit User Manual 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 ul well TMB substrate solution Take appropriate volume of TMB Substrate 100 ul 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 ul well and warm it up to room temperature before use After use discard remaining Stop Solution PROCEDURE Determine the number of wells and strips needed
4. p53 as a protein standard Starting with a 250 ng ul working stock of recombinant p53 make serial dilution in a range of 5 ng ul to 0 ng ul using Binding Buffer A without DTT Note the recommended dilution range is provided as guidance a broader range of values may be used Recombinant p53 solution Binding Buffer A p53 standard Final without DTT concentration volume B 25ulofA g l 50u D 25plofC ASQ 50W Wo Ong plank ow 10 ul 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 P53 positive control If quantitation is not necessary 10 ul of the 2 5 ng ul p53 protein standard can be used as the positive control 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 nuclear extract samples 1x Binding Buffer A with 2 mM DTT should be used For the recombinant p53 as protein standard and positive control 1x Binding Buffer A without DTT should be used 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
5. GeneCopoela i Expressway to Discovery TF Detect 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 2011 GeneCopoeia Inc TF Detect Human p53 Activity Assay Kit User Manual USER MANUAL TF Detect Human p53 Activity Assay Kit Introduction and Principle Kit Components and Storage Preparation Procedure References Appendix Limited Use License and Warranty 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 activity by failing to bind to the consensus DNA binding site TF Detect Human p53 Activity Assay kit enables fast and sensitive detection and quantification of p53 in a 96 well format Double stranded oligonucleotides containing a p53 consensus binding site are immobilized in a 96 well plate The p53 proteins present in nuclear extracts are capt
6. Store the unused strips at 4 C Mix all reagents thoroughly yet gently to avoid foaming before use Rinse the 96 well plate with approximately 200 yl 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 Add p53 samples to the wells as following Nuclear extract wells Add 50 ul of Binding Buffer A with DTT to the wells and then add 10 ul samples 2 50 ug 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 ul of p53 standard in duplicate to the standard wells and mix with 50 ul of Binding Buffer A without DTT Positive control wells if protein standard curve is not performed Add 10 ul of 2 5 ng ul p53 recombinant protein per well Add 50 ul of Binding Buffer A without DTT and mix well Blank wells Add 60 ul of Binding Buffer A without DTT 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 using 200ul well of 1X Wash Buffer by gently rocking it for one minute and then empty and tap the plate on absorbent pad or paper towel to remove excess buffer R
7. cement 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 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 GeneCopoeia Products are for Research Use Only Copyright 2011 GeneCopoeia Inc Trademarks GeneCopoeia TF Detect GeneCopoeia Inc TAKP53 052711
8. epeat the wash step twice Add 100 ul of 1 1000 diluted p53 primary antibody in 1X Binding Buffer B 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 with 200 ul well of 1X Wash Buffer by gently rocking it for one minute Then empty and tap the plate on absorbent pad or paper towel to remove excess buffer Repeat the wash step twice 10 11 Vi mae Oe Ae OnO 12 13 TF Detect Human p53 Activity Assay Kit User Manual Add 100 ul of 1 1000 diluted HRP conjugated antibody in 1X Binding Buffer B 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 with 200 ul well of 1X Wash Buffer by gently rocking it for one minute Then empty and tap the plate on absorbent pad or paper towel to remove excess buffer Repeat the wash step twice Add 100 ul of TMB Substrate Solution equilibrated to room temperature to each well including the blank wells and mix well Incubate the plate at room temperature for about 5 15 minutes Add 100 ul Stop Solution equilibrated to room temperature to each well including the blank wells and mix well Read the plate at 450nm within 5 minutes using a microwell plate reader Reference Hollstein
9. ivity Assay kit and a similar competitor product A human recombinant p53 protein was detected and quantified using both kits 0 0 78 1 56 3 13 625 125 25 50 P53 proteins ng Standard Curve of P53 Activity 3 Figure 2 Standard curve for p53 25 quantitation is generated using GeneCopoeia s purified p53 0 9839 recombinant protein included in the 1 5 4 kit The curve is provided for 1 demonstration only 0 5 0 0 20 40 60 P53 proteins ng P53 activity in MCF 7 Cells P53 activity in HEK293 Cells 3 2 2 8 Tt Q 1 1 fe 0 0 0 2 4 6 8 10 12 14 16 18 20 0 10 20 30 40 50 60 70 80 HEK293 Nuclear Lysates ug MCF 7 Nuclear Lysates ug 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 H202 for 3 hours before harvesting The cell nuclear extracts were prepared following the Preparation of Nuclear Extract protocol in the Appendix TF Detect Human p53 Activity Assay Kit User Manual Kit Components and Storage Cat No TAK P53 196 1 plate 96 reactions Components Quantity Storage temperature p53 antibody 15 ul 20 C Stable for at least 6 months HRP conjugated IgG 15 ul 20 C Stable for at least 6 months Recombinant p53 protein 20 ul 20 C Stable for at least 6 months 0 25yg ul Dithiothreitol DTT 1M TMB Substrate Solution 96 well p53 as
10. say plate 12 1 plate 4 C Stable for at least 6 months strips 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 ul to 1 000 ul adjustable single channel micropipettes with disposable tips 50 ul to 300 pl adjustable multichannel micropipette disposable tips and reservoir Micro plate reader capable of reading at 450 nm 620 nm as optional reference wave length 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 Reagent ma wi 1x Binding buffer A without DTT 1x Wash buffer 1x Binding Buffer A with DTT is used for the dilution and binding reaction of sample nuclear extracts 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 TF Detect Human p53 Activity Assay Kit User Manual Nuclear extract samples We recommend using 10 ul of nuclear extract at 0 2 5 ug ul for each sample well The total protein amount is 2 50 ug 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
11. ured by the immobilized oligonucleotides specifically and then detected by a p53 antibody and a HRP conjugated 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 e Sensitive Detects as little as 0 8 ng of human p53 protein per well and performs better than a similar competitor kit Fig 1 e HTS compatible Optimized for use on 96 well plate readers for high throughput screening assays Single strip 8 well assay can also be performed e Fast 3 5 hours from preparation to detection e 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 at p53 antibody 4 hour Wash 3X Add HRP conjugated IgG antibody 1 hour Wash 3X Colorimetric reaction Total 3 5 hours OD450 OD450 OD450 TF Detect Human p53 Activity Assay Kit User Manual Comparison of P53 Activity Assay Kits m GeneCopoeia Competitor Figure 1 Performance comparison between GeneCopoeia s TF Detect Human p53 Acit
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