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CY-7049

1. Etoposide treatment time hr Roa CY 7049 11 Version 140318 o c A Total p53 ELISA Kit gt O cA ycLex User s Manual A For Research Use Only Not for use in diagnostic procedures F References 3 Levine A J 1997 Cell 88 323 331 gt 2 Donehower L A Harvey M Slagle B L McArthur M J Montgomery C A Jr el J S and Bradley A 1992 Nature 356 215 221 3 Harvey M McArthur M J Montgomery C A Jr Bradley A and DonehoweQ y A 1993 FASEB J 7 938 943 4 Hollstein M Sidransky D Vogelstein B and Harris C C 1991 Science 253 5 Greenblatt M S Bennett W P Hollstein M and Harris C C 1994 Cancer 54 4855 4878 6 Chen H E Chang S Trub T and Neel B G 1996 Mol Cell Biol 16 3685 697 7 Honda R Tanaka H and Yasuda H 1997 FEBS Lett 420 25 27 Yon 8 9 1 Kubbutat M H Jones S N and Vousden K H 1997 Nature 387 29 Haupt Y Maya R Kazaz A and Oren M 1997 Nature 387 DOA 0 McCoy M A Gesell J J Senior M M and Wyss D F 2003 PRS Ni 1645 1648 11 Barak Y Juven T Haffner R and Oren M 1993 EMBO J 1246 1 468 12 Appella E and Anderson C W 2001 Eur J Biochem 268 4 2772 13 Wahl G M and Carr A M 2001 Nat Cell Biol 3 E277 14 Buschmann T Potapova O Bar Shira A Ivanov V N Fuchs S Y Henderson S Fried V A
2. N e Upon receipt store components at 4 C e Don t expose reagents to excessive light o gL eA Roa CY 7049 1 Version 140318 o c Total p53 ELISA Kit P 4 r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Introduction 3 p53 is a short lived non abundant protein that regulates the response of cells to DNA dam n part through transcriptional activation of genes involved in cell cycle control DNA repair and apQptosis 1 p53 is a tumor suppressor protein consequently mice in which the p53 gene has been disrapie d develop tumors with high frequency 2 3 and deletions or mutations in the p53 gene are prevalegfyin a majority of human cancers 4 5 The protein level and activity of p53 are regulated and this i omplished by MDM2 murine double minute 2 6 10 The MDM2 gene is induced by p53 pipu prevents apoptosis by inhibiting p53 activity and promoting its degradation 6 9 11 In response to DNA damage the p53 protein is phosphorylated on each of the seven serines and one threonine the in the first 50 amino acids of its N terminal transactivation domn as well as at several sites in its C terminal tetramerization regulatory domain 12 13 As a ragy ption factor p53 induces or represses several genes that regulate cell cycle arrest DNA repair or apoptosis including p21 WAFI MDM2 GADD45 p53R2 and p53AIP1 Recent studies suggest thi ecific p53 phosph
3. Minamoto T Alarcon Vargas D Pincus M R Gaar Ry A Holbrook N J Shiloh Y and Ronai Z 2001 Mol Cell Biol 21 2743 2754 15 Dumaz N and Meek D W 1999 EMBO J 18 700337010 16 Jabbur J R Huang P and Zhang W 2000 Onc ggne 19 6203 6208 17 Oda K Arakawa H Tanaka T Matsuda K nikawa C Mori T Nishimori H Tamai K Tokino T Nakamura Y and Taya Y 2000 102 849 862 atl Acad Sci U S A 100 Related Products y CycLex Total p53 ELISA Kit Cat 49 CycLex Phospho p53 S46 ELISA Kite Cat CY 7050 CycLex Phospho p53 S392 ELISAQSit Cat CY 7051 Anti Phospho p53 S46 a tai antibody Cat CY M1022 amp PRODUCED BY A CycLex Co Ltd N 1063 103 Terasaw Ina Nagano 396 999 Japan Fax 481 265 264618 e mail a Sere wep URL mie w cyclex co jp CycL AXCircuLex products are supplied for research use only CycLex CircuLex products and comp pents thereof may not be resold modified for resale or used to manufacture commercial pro cts without prior written approval from CycLex Co Ltd To inquire about licensing for s commercial use please contact us via email Roa CY 7049 12 Version 140318 o c
4. O L EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE METERS BEING INVESTIGATED AND MUST BE DETERMINED BY THE DIVIDUAL USER NO WARRANTY OR GUARANTEE OF PERFORMANCE USING THESE PROCEDURES IS MADE OR IMPLIED 2 Roa CY 7049 7 Version 140318 o AO Total p53 ELISA Kit Pa P ycLex User s Manual C ELISA 1 2 X 3 Nn oOo 9 For Research Use Only Not for use in diagnostic procedures Remove the appropriate number of microtiter wells from the foil pouch and place them intedhe well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C Ww Dilute the lysate 1 10 with Dilution Buffer e g 30 uL sample 270 uL Dilution Bulg 1 10 sample dilution has been found to be satisfactory higher dilutions such as 0 or 1 40 may be Lysates prepared in Cell Extraction Buffer must be diluted 1 10 or greater in Digs te While a optimal The dilution chosen should be optimized for each experimental ISTEN a Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted reg in duplicates into the appropriate wells N Incubate the plate at room temperature ca 25 C for 1 hour shah at ca 300 rpm on an orbital microplate shaker Wash 4 times by filling each well with Wash Buffer 350 using a squirt bottle multi channel pipette manifold dispenser or microplate washer Add 100 uL of Primary Antibody Solution into each wg Incubate
5. Total p53 ELISA Kit re lt QR P 4 r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Troubleshooting 3 1 The Human p53 Standard should be run in duplicate using the protocol described in th Mailed Protocol Incubation times or temperatures significantly different from those specifiedNmay give erroneous results 2 Poor duplicates accompanied by elevated values for wells containing no sample i te insufficient washing If all instructions in the Detailed Protocol were followed accurately s esults indicate a need for washer maintenance Q 3 Overall low signal may indicate that desiccation of the plate has occurred been the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add S te Reagent immediately after wash Reagent Stability 3 All of the reagents included in the CycLex Research Produc cLex Total p53 ELISA Kit have been tested for stability Reagents should not be used beyond th ted expiration date Upon receipt kit reagents should be stored at 4 C except the reconstituted p53 Standard must be stored at below 70 C Coated assay plates should be stored in the original foil sealed by the zip lock and containing a desiccant pack R Assay Characteristics R S Ww 1 Sensitivity The limit of detection defined as such a cOfAgentration of p53 giving absorbance higher than mean absorbance of blank plus three standard deyfations of the absorbance
6. of blank A blank 3 SD blank is better than 1 335 units ml of sample KX Dilution Buffer is pipetted into blank Wis Eighty assays were evaluated a 1 637 units mL The mean MD standard deviations to the m the corresponding concentra e minimum detectable dose MDD of p53 ranged from 0 926 as 1 335 units mL The MDD was determined by adding three tical density value of twenty zero standard replicates and calculating 2 Specificity N The antibodies in p53 ELISA kit are highly specific of human p53 with no detectable cross reactivity to and rat p53 3 Linearity To assess t earity of the assay samples containing and or spiked with high concentrations of p53 were seriall uted with the Dilution Buffer to produce samples with values within the dynamic range y Total p53 ELISA Kit 9 f ycLex User s Manual 3 For Research Use Only Not for use in diagnostic procedures 4 Example of Test Results 3 Fig l Typical standard of p53 mS Standard curve Y 7 S 2 0 1 5 1 0 Pa 0 5 F 0 20 40 60 80 100 Total p53 U ml A450 0 0 Ww Fig 2 Accumulation of p53 in breast cancer ee MCF 7 that expresses wild type p53 after treatment with 100 uM Etoposide for indicated tipne Total p53 was measured by CycLex Total p53 ELISA kit 2 K Total p53 ELISA MCE 7 treated with 100 uM Etoposide for indicated time 2 5 p A450 0 0 h 0 1 2 3 4 5 6 7
7. the Stop Solution Roa CY 7049 8 Version 140318 o AO A S Total p53 ELISA Kit 9 A ycLex User s Manual 3 For Research Use Only Not for use in diagnostic procedures 4 gt O Note 1 Complete removal of liquid at each step is essential to good performance After the lastNysh remove any remaining Wash Buffer by aspirating or decanting Invert the plate olot it against clean paper towels Wh Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 4its for the blank zero concentration or 2 5 units for the highest standard oonan The plate should be monitored at 5 minute intervals for approximately 30 minutes Note 3 If the microplate reader is not capable of reading absorbance greater than t sorbance of the highest standard perform a second reading at 405 nm A new stand rve constructed using the values measured at 405 nm is used to determine p53 me tration of off scale samples The readings at 405 nm should not replace the on scale readag amp at 450 nm Calculations Average the duplicate readings for each standard control and sama and subtract the average zero standard optical density Plot the optical density for the standard ersus the concentration of the standards and draw the best curve The data can be eming ps log log paper and regression analysis may be applied to the log transformation To determin p53 concentration of each sample first find the absorbance va
8. d 100 uL oitir WS Woon at 450 nm O nw Measure a a 2 Roa CY 7049 3 Version 140318 o c A Q Total p53 ELISA Kit re lt QR P 4 cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Materials Provided 3 All samples and standards should be assayed in duplicate The following components are su d and are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells ing foil zip lock bag with a desiccant pack Wells are coated with anti human p53 antibody as a care ay 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 2 Cell Extraction Buffer One bottle containing 20 mL of 1X buffer y Dilution Buffer One bottle containing 50 mL each of 1X buffer use for Q dilution Ready to use p53 Standard One vials each vial contains 200 units of lyophilized p Primary Antibody Solution Anti p53 Monoclonal Antibody DAN One vial containing 12 mL of Anti p53 Monoclonal Antibody Ready to use Secondary Antibody Solution HRP conjugated Anti M IgG One vial containing 12 mL of HRP horseradish peroxidase conjugated anti mouse IgG ody Ready to use Substrate Reagent One bottle containing 20 mL of Oj chromogenic substrate tetra methylbenzidine TMB Ready to use ny Ww Stop Solution One bottle supplied ready to use ptaining 20 mL of 1 N H2501 n WY Materials Required b
9. ic procedures 4 Detailed Protocol 3 The CycLex Research Product CycLex Total p53 ELISA Kit is provided with removabl ips of wells so the assay can be carried out on separate occasions using only the number of strips ired for the particular determination Since experimental conditions may vary an aliquot of the ere within the kit should be included in each assay as a calibrator Disposable pipette t y and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents amples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Asgevreagents are supplied ready to use with the exception of 10X Wash Buffer Cell Extraction Bui d p53 Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water ddH2O Mix well Store at 4 C for tw eeks or 20 C for long term storage Q 2 Prepare a working solution of Cell Extraction Buffer by addin 0 uL of Protease inhibitor cocktail Sigma Cat P 2714 to 5 mL of Cell Extraction Buffer Mik Well 3 Reconstitute p53 Standard with 1 mL of ddH20 The c tntration of the p53 in vial should be 200 units mL which is referred as a Master Standard of nag Prepare Standard Solutions as follows CA Use the Master Standard to produce a cay series below Mix each tube thoroughly before the next transfer The 100 uni
10. ided with the kit e Rinse all detergent residue from glassware e Use deionized water of the highest quality RU S Y A N e Do not mix reagents from different kits amp The buffers and reagents in this kit may contain preservatives ier chemicals Care should be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents RZ e Do not smoke eat or drink when performing the say or in areas where samples or reagents are handled A e Dispose of tetra methylbenzidine TMB contei g solutions in compliance with local regulations e Avoid contact with the acidic Stop Soluig Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection whet Fandting immunodiagnostic materials and samples of human origin and these reagents In case ontact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and se gmedical attention when necessary Biological samples may Pacer ssaiel with infectious agents Do not ingest expose to open wounds or breathe aerosdls Wear protective gloves and dispose of biological samples properly handli p Solution O nw a Qe amp eo Roa CY 7049 5 Version 140318 e CAUTION handia id is a strong acid Wear disposable gloves and eye protection when o AO A Q Total p53 ELISA Kit re lt QR P 4 r ycLex User s Manual For Research Use Only Not for use in diagnost
11. in at captured during the first incubation After removal of excegg letection antibody followed by binding with horseradish peroxidase conjugated anti mouse IgG h then catalyzes the conversion of the chromogenic substrate tetra methylbenzidine TM om a colorless solution to a blue solution or yellow after the addition of stopping reagent The colexis quantitated by spectrophotometry and reflects the relative amount of p53 present in the original s en oO B O RS Q e Roa CY 7049 2 Version 140318 o AO wy Q Total p53 ELISA Kit re A Q ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Summary of Procedure l Culture cells in culture flask or dish at 50 70 confluency SS Incubate O N at 37 C in CO incubator K Add appropriate amount of the drug for induction of p53 accumulation amp 4 Incubate appropriate time at 37 C in CO2 incubator g Harvest the cells by scraping and centrifugation 4 F Make cell lysate by adding extraction buffer and centrifugati S i gt Add 100 uL of diluted cell lysate to the wells J Incubate for 1 hour at room temp N Wash the wells j o Add 100 uL of Primary Antibody Solution ips monoclonal antibody 4 Incubate for 1hour at roq temp Wash the wells SS Add 100 uL of Secondary Antibodg olution HRP conjugated anti mouse IgG antibody 4 Incubate for Thur at room temp Wash the wells x Add 100 uL of SubstghCReagen t A Ad
12. lue on the y axis and extend a hogizontal line to the standard curve At the point of intersection extend a vertical line to the x axis and the corresponding p53 concentration If the samples have been diluted the concentration read frog standard curve must be multiplied by the dilution factor A The dose response curve of this assay fits be a sigmoidal 5 parameter logistic equation The results of unknown samples can be calculated With any computer program having a 5 parameter logistic function It is important to make an qp opriate mathematical adjustment to accommodate for the dilution factor 4 B Most microtiter plate readers perform a atic calculations of analyte concentration The calibration curve is constructed by plotting t bsorbance Y of calibrators versus log of the known concentration X of calibrators wsimg the four parameter function Alternatively the logit log function can be used to linearize t libration curve i e logit of absorbance Y is plotted versus log of the known concentration X ibrators Measurement Rage The measurement ra 56 units mL to 100 units mL Any sample reading higher than the highest standard should be ina ith Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken into a o tion in calculating the human p53 concentration F amp gt o G oe Roa CY 7049 9 Version 140318 lt C Roa CY 7049 10 Version 140318 amp c A Q
13. orylation events are important for the activation or repression of specific promot 14 17 It has been shown that the proline rich domain residues 64 9 sand a transcriptional activation domain residues 43 63 of p53 have each been suggested to be ssary for mediation of apoptosis because deletion of either of these two domains abolishes this a y It was found that phosphorylation of Ser46 is induced after DNA damage in vivo Particularly phosphorylation of Ser46 was observed only at the late stage after DNA damage by several different aget compared to Ser15 or Ser20 Moreover upon severe DNA damage by UV Ser46 on p53 is phosp lated and apoptosis is induced In addition substitution of Ser46 to Alanine inhibits the ability of pS Jto induce apoptosis but not G1 arrest These results suggested that phosphorylation of Ser46 is involy3d in induction of p53 dependent apoptosis Principle of the Assay The CycLex Research CycLex Total p53 EL A Kit is a solid phase sandwich ELISA An antibody specific human p53 has been coated onto ells of the microtiter strips provided Samples including a standard containing p53 control ri and unknowns are pipetted into these wells During the first incubation p53 protein binds to the ture antibody on the well After washing mouse monoclonal antibody specific p53 as a detection g pfibody is added to the wells During the second incubation this antibody serves as a detector by ing to the immobilized p53 prote
14. p Total p53 ELISA Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Total Human p53 O g P N CycLex Total p53 ELISA Kit Q Cat CY 7049 amp Mri S CISC ocasacarsasnteandadciotaienceciesamkectond 1 Meg SOOO Se T 1 oO TCO G HOM pcp 5385s ddoweacasomneuarniuene 2 aS Principle Of the Assay 2 3 XN Materials Provided scisccsessscccsstsesssssovacesecsvess 4 O Materials Required but not Provided 4 gt Precautions and Recommendations 5 N Detailed Proto jaicassescessvetavesssncsessuaniooensees 6 9 MAL CRL ALI ONS sich scavantunisedacnsansgseavncons risinn 9 re Measurement Range 9 amp Prouble shoots 5 3 csnssshucevedesnsdatonamessiecneee 10 Ka Reagent Stability 10 gt Assay Characteristics ccccccsscessseeeeones 10 Example of Test Results eeeeeeeeeeee TS REPCRENCES ssai csacaisiasssaunsasscvetosersusvavauteeasareuace 6 Related Proguctssissiseseccsevssessasceeasteuaveneas W Intended Use sO The CycLex Research Product CycLetotal p53 ELISA Kit is designed to detect and quantify the level of total human p53 in cell lysategfince the amino acid sequence p53 is not conserved in mouse and rat p53 this ELISA kit can not be Bf for mouse and rat cells This assay is intended for the detection of total human p53 in cell lysate Qy This assay kit is for researagQnt only and not for use in diagnostic or therapeutic procedures
15. ral cell li sers should optimize the cell extraction procedure for their own applications Y 1 Collect cells in PBS by centrifugation non adherent cells of amp scraping from culture flasks adherent cells 2 Wash cells twice with cold PBS KG 3 Remove and discard the supernatant and collect t pellet At this point the cell pellet can be frozen at b 70 C and lyse at a later date 4 Lyse the cell pellet in 0 5 mL of Cell Exton Buffer for 30 minutes on ice with vortexing at 10 minute intervals Py To get a rough idea you could adj Se cell concentration to around 2 x 10 cells mL Resulting protein concentration of the cell lyste should be 2 4 mg mL using this Cell Extraction Buffer The volume of Cell aioe Buffer depends on the cell number in cell pellet and phosphorylation level of p53 Kr example 1 x 10 MCF 7 cells in 10 cm dish can be extracted in 0 5 mL of Cell Extractio er Under these conditions the protein concentration should be 2 4 mg mL and use of 10 2 of the clarified cell extract diluted to a volume of 100 ul well in Dilution Buffer is sufficient for etection of phospho p53 S46 5 Transfer the lysate tagnicrocentrfuge tubes and centrifuge at 15 000 rpm for 10 minutes at 4 C 6 Aliquot the clear lysate to clean microfuge tubes These samples are ready for assay The lysates can be stored at ni 70 C Avoid multiple freeze thaw cycles NOTE T O BOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE
16. the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker Wash 4 times by filling each well with Wasepuffer 350 uL using a squirt bottle multi channel pipette manifold dispenser or microplate wa hgr Add 100 uL of Secondary Antibody Sion into each well 10 Incubate the plate at room tem erature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker Q pipette manifold dispens icroplate washer 11 Wash 4 times by filling app with Wash Buffer 350 uL using a squirt bottle multi channel 12 Add 100 uL of Substrate Reagent Avoid exposing the microtiter plate to direct sunlight Covering the plate with e g um foil is recommended Return Substrate A to 4 C immediately after the necessary volume i femoved 13 Incubate the plate at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital micr e shaker The incubation time may be extended up to 30 minutes if the reaction temperatu elow than 20 C 14 Add wy of Stop Solution to each well in the same order as the previously added Substrate Re 15 M asure absorbance in each well using a spectrophotometric microplate reader at dual wavelengths Qf 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate t 450 nm if only a single wavelength can be used Wells must be read within 30 minutes of adding N S amp
17. ts mL standayd Std 1 serves as the highest standard The Dilution Buffer serves as the zero standard Blank gt Volume of sa Dilution Buffer Concentration Std 1 250 uL of Master es 250 uL 100 units mL Std 2 250 uL of Std 1 units mL 250 uL 50 units mL Std 3 250 uL of Std 0 units mL 250 uL 25 units mL Std 4 250 uL of S 25 units mL 250 uL 12 5 units mL Std 5 250 uL 4 12 5 units mL 250 uL 6 25 units mL Std 6 250 HAO td 5 6 25 units mL 250 uL 3 13 units mL Std 7 25 of Std 6 3 13 units mL 250 uL 1 56 units mL Blank 250 uL 0 units mL before d pensing Unused portions of Standards should be aliquoted and stored at below 70 C Note Do not a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer bi Oe Avoid multiple freeze and thaw cycles amp gt o G oe Roa CY 7049 6 Version 140318 o c SS Total p53 ELISA Kit re lt Q Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures S gt Assay Procedure l A Treatment of Cells with compounds S 1 Plate adherent cells or non adherent cells in culture flasks at 50 70 confluency KS 2 Incubate the culture flasks at 37 C over night in CO2 incubator g 3 Add appropriate amount of test compounds to each flask 2 4 Incubate the culture flasks at 37 C for appropriate time B Cell Extraction y D gt Note This protocol has been successfully applied to seve
18. ut not Repvided e Protease inhibitor cocktail ex Sing Cat P 2714 reconstituted according to manufacturer s guideline Add 250 uL per 5 mL xtraction Buffer Orbital microplate shaker e Pipettors 2 20 uL 20 200 uL 00 1000 uL precision pipettors with disposable tips e Precision repeating pipetton e Microcentrifuge and tub sample preparation e Vortex mixer e Microplate washer optitgnal Manual washing is possible but not preferable e Plate reader capable easuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavele of 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelengt 450 nm which will give a somewhat higher reading e Software package facilitating data generation and analysis optional e 500 or 1000 m amp gpraduated cylinder e Reagent res irs Deionize Kater of the highest quality aper towels Roa CY 7049 4 Version 140318 o c S Total p53 ELISA Kit 9 ycLex User s Manual 3 For Research Use Only Not for use in diagnostic procedures 4 Precautions and Recommendations 3 e Allow all the components to come to room temperature before use we All microplate strips that are not immediately required should be returned to the zip lock Na which must be carefully resealed to avoid moisture absorption g e Do not use kit components beyond the indicated kit expiration date Yon e Use only the microtiter wells prov

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