CY-8096 Human Hsp27 ELISA Kit
Contents
1. CY 8096 9 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting j The Standard Solutions should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenance 3 Overall low signal may indicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Reagent Stability All of the reagents included in the CycLex Research Product CircuLe Human Hsp27 ELISA Kit have been tested for stability Reagents should not be used beyondgthe stated expiration date Upon receipt kit reagents should be stored at 4 C except the reconstituted Hsp27 Standard must be stored at below 70 C The Microplate should be stored in the originaldfoi bag sealed by the zip lock and containing a desiccant pack Cat CY 8096 10 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Severa2. 53 629 635 Related Products CircuLex Human Fibulin 1 ELISA Kit Cat CY 8094 CircuLex Human Chitotriosidase ELISA Kit Cat CY 8074 CircuLex Human YKL 39 ELISA Kit Cat CY 8087 CircuLex Human YKL 40 ELISA Kit Cat CY 8088 CircuLex Human DJ 1 PARK7 ELISA Kit Cat CY 9050 CircuLex Human S100A12 ELISA Kit Cat CY 8058 CircuLex Human RBP4 ELISA Kit Cat CY 8072 CircuLex Human Hsp27 ELISA Kit Cat CY 8096 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyclex co jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 8096 19 Version 140530
3. 60 ng mL Std 1 60 uL of 10X Std 1 60 ng mL 540 uL 6 000 pg mL Std 2 300 uL of Std 1 6 000 pg mL 300 uL 3 000 pg mL Std 3 300 uL of Std 2 3 000 pe mL 300 uL 1 500 pg mL Std 4 300 uL of Std 3 4300 pg mL 300 uL 750 pg mL Std 5 300 uL of Std 4 750 pg mL 300 uL 375 pg mL Std 6 300 uL of Std 5 875 pg mL 300 uL 188 pg mL Std 7 300 uL of Std 6 188 pg mL 300 uL 94 pg mL Blank 300 uL 0 pg mL Note Do not use a Repeating pipette Change tips for every dilution Unused portions of Master Standard should b aliguoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation e Optimal dilution of Cell lysates for measurement of Hsp27 concentration depends on cell lines and numbers require neat to appropriate dilution e Serum and plasma samples require a 25 50 fold dilution depending on Hsp27 concentration in samples Cat CY 8096 7 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure j Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C N Dilute samples with Dilution Buffer See Sample Preparation above W Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in duplicates into the
4. ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the human Hsp27 Standard within the kit should be included in each assay as a calibratof Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r agents are supplied ready to use with the exception of 10X Wash Buffer and Human Hsp27 Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer provided to 900 mL of deionized distilled water ddH O Mix well 2 Reconstitute Human Hsp27 Standard with 1 0 mL of Dilution Buffer The concentration of the Hsp27 in vial should be 1 000 ng mL which is referred as a Master Standard of Hsp27 Prepare Standard Solutions as follows Use the Master Standard 1 000 ng mL to produce 10X Std 1 60 ng mL and make a dilution series below Mix each tube thoroughly before the diext transfer The 6 000 pg mL standard Std 1 serves as the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration 10X Std 1 60 uL of Master Standard 1 000ng mL 940 uL
5. 2 Hsp27 can redistribute to the nucleus from the cytoplasm in esponse to stress where it may function to stabilize DNA and or the nuclear membrane Hsp27 was reported to be associated with poor prognosis in gastri liver and prostate carcinoma and osteosarcomas 13 14 However other study showed that Hsp27 was not detectable in metastatic tumors and could be found only in samples derived from non metastatic tumors 15 It was reported that the concentration of Hsp27 in serum from pancreatic tumor patients was found to be significantly higher than in serum from healthy controls 16 Principle of the Assay The CycLex Research Product CircuLex Human Hsp27 ELISA Kit employs the quantitative sandwich enzyme immunoassay technique An antibody specific for human Hsp27 is pre coated onto a microplate Standards and samples are pipetted imto the wells and the immobilized antibody binds any human Hsp27 present After washing away amy Unbound substances an HRP conjugated antibody specific for human Hsp27 is added to the wells Following a wash to remove any unbound antibody HRP conjugate the remaining conjugate is allowed to react with the substrate H2O2 tetramethylbenzidine The reaction is stopped by addition ofsacidies olution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to the concentration of human Hsp27 A standard curve is constructed by plotting absorbance values versus human Hsp27 concent
6. Hsp27 ELISA Kit CircuLex Users Manual 4 For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig l Human Hsp27 concentration after heat shock treatment determined by ELISA and We blotting Hsp27 concentration in lysates of HeLa cell after heat shock treatment for indicated times 1 2 EE EEN D z a c oe ie ea es mmm eee i ee x oh 6 je SS SS SS Se eee Se BR ere aa amp g S 0 4 F ptt TEH EO Ee se a 2 02 EF oe PES aS ia ie as ER 0 0 0 5 15 30 45 60 120 Heat shock treatment time minute Rd Heat s treatment time S S S ed ed E a pn Do A en kDa R 66 45 C CY 8096 16 Version 140530 Human Hsp27 ELISA Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures Fig 2 Hsp27 levels in biological fluids of healthy volunteers a Hsp27 levels in sera from 28 healthy volunteers Human Hsp27 conc n 28 jo Max 860 Min td Serum Hsp27 levels in 28 healthy volunteers 100 e 80 prennan ee Sea e S ON 3 60 F oles S S e a 8 40 er a 8 E 8 6 20 EE RE 0 Ld sample Hsp27 conc ng ml 1 127 3 2 171 4 C CY 8096 17 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References EN Clausen T So
7. Human Hsp27 ELISA Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human Hsp27 Ad CircuLex Human Hsp27 ELISA Kit AN Cat CY 8096 COS Intended Use ee ee ee RR Re 1 EASE OE N eN NE 1 IntroducHON esse ees see Re RA 2 Qy Principle of the ASSAY snoesig ses seke ER se 2 3 Materials Provided ees ee ee 3 Materials Required but not Provided 4 Precautions and Recommendation 5 Sample Collection and Storage esse ses 6 Detailed Protocol i iese sesse ee ee 7 8 Calculations cccccccecessceserseeeee cceenseteeees 9 Measurement Range sees sees ee ee ee 9 Troubleshooting essens see ese ed ak dd Ge AR 10 Reagent StDIEY ees ora acasaansranrecasunerdcotynirss 10 Sample PreparatiOn esse ses sn se Es Ed 11 Assay Charaoteristies sesse see sed Eg SR Ee Example of Test Results sesse seer gese gede Reference RA RA Re Re 18 Related ProductS ees esse ee ee Rg 19 Intended Use The CycLex Research Product Cire an Hsp27 ELISA Kit is used for the quantitative measurement of human Hsp27 in cell lys an serum and other biological media This assay kit is for research use d not for use in diagnostic or therapeutic procedures Storage e Upon receipt store all con at 4 C e Don t expose reagents to ex e light C CY 8096 1 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual For Research Use Only
8. Not for use in diagnostic procedures Introduction Hsp27 is an abundant and ubiquitous family of small HSP heat shock protein that acts asgan ATP independent chaperone and mainly localized in the cytosol 1 Small HSPs are potent mediator of protein folding and also involved in architecture of cytoskeleton cell migration cell growth differentiation and tumor progression 2 5 Hsp27 is also involved in the apoptotic signaling pathway because it interferes with the activation of cytochrome c Apaf 1 dATP complex thereby inhibiting the activation of procaspase 9 6 The basic structure of most small HSPs is a homologous and highly conserved amino acid sequence with an alpha crystallin domain at the C terminus and the WD EPF domain at the N terminus Hsp27 exists as both large oligomers that are proposed to have chaperone like activity and a smaller oligomers that bind and cap barbed end microfilaments and stabilize them 7 The up regulation of Hsp27 correlates with the rate of phosphorylation and with an increase of large oligomers High levels of Hsp27 have been observed in many cancer cells including breast carcinoma 8 9 ovarian cancer 10 and rectal cancer 11 compared to normal cells in which expression is undetectable or relatively low 2 Hsp27 is expressed constitutively in many tissues and its expression is increased to high levels after various types of stress including elevated temperatures toxic metals drugs and oxidants 1
9. appropriate wells A Incubate the wells at room temperature ca 25 C for 1 hour shaking at ca 30 TPmM on an orbital microplate shaker Nn Wash 4 times by filling each well with Wash Buffer 350 uL using a sguirtbottle multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each well Incubate the wells at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker OO Wash 4 times by filling each well with Wash Buffer 350 ul using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent to each well Avoid exposing the microtiter plate to direct sunlight Covering the wells with e g aluminum foil ig recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the wells at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation ime may be extended up to 30 minutes if the reaction temperature is below 20 C 11 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Measure absorbance in each well using aspectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a si
10. f chaperones in ten human tumor cell lines Proteome Ser 2004 2 8 10 Langdon SP Rabiasz GJ Hirst GL King RJ Hawkins RA Smyth JF Miller WR Expression of the heat shock protein HSP27 in human oyaryan cancer Clin Cancer Res 1995 1 1603 9 11 Tweedle EM Khattak I Ang CW eb al Low molecular weight heat shock protein HSP27 is a prognostic indicator in rectal caneer but not colon cancer Gut 2010 59 1501 10 12 Hickey E Brandon S E Potter R Stein G Stein J and Weber L A Nucleic Acids Res 1986 14 4127 4145 13 Garrido C Schmitt E ande Vahsen N Parcellier A Kroemer G HSP27 and HSP70 potentially oncogenic apoptosis inhibitors Cell Cycle 2003 2 579 84 14 Ciocca DR Calderwood SK Heat shock proteins in cancer diagnostic prognostic predictive and treatment implications Cell Stress Chaperones 2005 10 86 103 15 Chen J Kahne T Rocken C Gotze T Yu J Sung JJ et al Proteome analysis of gastric cancer metastasis by two dimensional gel electrophoresis and matrix assisted laser desorption iomizationmass spectrometry for identification of metastasis related proteins J Proteome Res 2004 3 1009 16 Cat CY 8096 18 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 16 Melle C Ernst G Escher N et al Identification of HSP27 amp DJ 1 as a Potential Pancreas Carcinoma Serum marker Clin Chem 2007
11. l extraction methods can be used for measurement cellular total Hsp27 The following protocol has been shown to work with a number of different cell lines and is provided as an example of suitable methods It is strongly advised that the user always perform an initial experiment to determine the proper dilution to be used in subsequent experiments This need not be any more than a single time point assay using serial dilutions of the cell lysate One eight well strip of the microplate should be sufficient for this initial experiment All steps of cell lysate preparation should be performed at 4 C and recovered cell lysates should be kept at below 70 C Preparation of Cell Lysate A Preparation of Cell Lysis Buffer 20 mM Tris HCl pH 7 5 250 mM NaCl 10 glycerol 0 1 NP 40 1 mMAEDTA 1 mM EGTA 0 2 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin 0 2 mM DTT B Preparation of poly l lysine coated plate Coat the plate with 25 ug mL poly l lysine PLL in PBS for 4 12sh at 37 C Subsequently go to a washing step with PBS C Treatment of Cells 1 Plate adherent cells in PLL coated 24 well plate at around 5 X 10 ells well 2 Incubate the culture dish at 37 C for 12 14 hours i CO incubator 3 Treat the cell with a variety of stimuli for appropriate time e g heat shock or test compounds and vehicle for test compound to each well 4 Incubate the culture dish at 37 C for 6 8 hours of appropriate time for induction of Hsp27 expre
12. lection and Storage Cell lysate Prepare cell lysates Assay immediately or store the samples on ice for a few hours b assaying Aliquots of the samples may also be stored at below 70 C for extended periods of time Avo repeated freeze thaw cycles Serum Use a serum separator tube and allow samples to clot for 60 30 minutes samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or sto ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Naz as the anticoagulant If possible colle e plasma into a mixture of EDTA Naz and Futhan5 to stabilize the sample against spontane in o complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x y immediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma a e stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay Other biological samples Remove any particulates by centrifugati ssay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw s C CY 8096 6 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Human Hsp27
13. ngle wayelength can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against Clean pap r towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 20 units for the blank Zero concentrations or 3 0 units for the highest standard concentration The plate should be monitored at 5 minute intervals for approximately 30 minutes Note 3 Jf the micfoplate reader is not capable of reading absorbance greater than the absorbance of the highestystandard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine Hsp27 concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Cat CY 8096 8 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations Average the duplicate readings for each Standard Solution control and sample and subtractathe average zero standard optical density Plot the optical density for the standards versus the concentration of the standards and draw the best curve The data can be linearized by using log log paper and regression analysis may be applied to the log transfo
14. ration date e Use only the microtiter wells provided with the kit e Rinse all detergent residues from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or othetchemi als Care should be taken to avoid direct contact with these reagents e Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containifig solutions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection when handling immunodiagnostic materials and samples of human origin and these reagents In case of contaetywith the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly e CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8096 5 Version 140530 Human Hsp27 ELISA Kit CircuLex Users Manual 4 For Research Use Only Not for use in diagnostic procedures Sample Col
15. rations of calibrators and concentrations of unknowngsamplesjare determined using this standard curve Cat CY 8096 2 Version 140530 Human Hsp27 ELISA Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells 4 Incubate for 1 hour at room temp Wash the wells O N Add 100 uL of HRP conjugated anti human Hsp27 antibody 4 Incubate for 1 hour at room temp Wash the wells Add 100 uL of Substrate Reagent 5 Add 100 uL of Stop Solution O t Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in are sufficient for the one 96 well microplate kit e The following components are supplied and Microplate One microplate supplied ready t bag with a desiccant pack Wells are coated 10X Wash Buffer One bottle ek of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containi f 1X buffer use for sample dilution Ready to use Human Hsp27 Standard One SS 1 ug of lyophilized recombinant human Hsp27 Sue with 96 wells 12 strips of 8 wells in a foil zip lock human Hsp27 antibody as a capture antibody HRP conjugated Detectio y One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti human Hs y Ready to use Substrate Reagent containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready tofuse Stop Solution O
16. rmation To determine the himan Hsp27 concentration of each sample first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the point of intersection extend a vertical line to the x axis and read the corresponding human Hsp27 concentration If the samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal 4 parameter logistic equation The results of unknown samples can be calculated with any computer programghaving a 4 parameter logistic function It is important to make an appropriate mathematical adjustment to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curve is constructed by plotting the absorbance Y of calibrators versus log of the known concentration X of calibrators using the 4 parameter function Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance X is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 94 pg mL to 6 000 pg mL Anyf ample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken into consideration in calculating the human Hsp27 concentration Cat
17. sssion D Cell Extraction Note This protocol has been successfullyapplied to HeLa cell line Users should optimize the cell extraction procedure for th ir wn applications 1 Wash cells three times with ieescold PBS Remove any remaining PBS by decanting Invert the plate and blot it against clean paper fowels At this point the cells in the plate can be frozen at below 70 C and lysed at a later date 2 Lyse the cells by adding 0 2 mL of Cell Lysis Buffer for 60 90 minutes at 4 C with rotating at ca 200 rpm by an orbital microplate shaker To get a rough idea you could adjust the cell concentration to around 2 5 3 0 x 10 cells mL in Cell Lysis BuffersResulting protein concentration of the HeLa cell lysate should be 0 4 0 5 mg mL using this procedure The appropriate volume of Cell Lysis Buffer depends on the cell line the cell number and the amount oftotal Hsp27 For example 5 x 10 HeLa cells can be lysed in 0 2 mL of Cell Lysis Buffer Cat CY 8096 11 Version 140530 P Di Human Hsp27 ELISA Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Centrifuge at 3 500 rpm for 15 minutes at 4 C using a microplate bucket or transfer the cell lysates to microcentrifuge tubes and centrifuge at 15 000 rpm for 5 minutes at 4 C 4 Transfer the clear cell lysates to new 24 well plate or clean microcentrifuge tubes Dilute thes lysates 5 10 times with Dilution Buffer 100 uL of
18. these diluted cell lysates are ready for assayf Go to the section Standard Assay Procedure for Human Hsp27 at page 8 Typical data using this protocol are shown in Fig page 16 The cell lysates can be stored at below 70 C Avoid multiple freeze thaw cycles After thaw the cell lysates centrifuge at 15 000 rpm for 5 minutes at 4 C again since the cell lysates sh e clear of any sediments or particulate matter NOTE Although we suggest to conduct experiments as outlined above imal experimental conditions will vary depending on the parameters being investigated st be determined by the individual user NO WARRANTY OR GUARAN ERFORMANCE USING THESE PROCEDURES IS MADE OR IMPLIED C CY 8096 12 Version 140530 Human Hsp27 ELISA Kit CircuLex Users Manual 4 For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of human Hsp27 giving absorbance high mean absorbance of blank plus three standard deviations of the absorbance of blank A bl D blank is better than 93 pg mL of sample Dilution Buffer is pipetted into blank wells Typical Standard Curve 1 8 p Q 16 Standard curve 9 A450 0 1 000 2 000 3 000 4 000 5 000 6 000 Hsp27 concentration pg ml C CY 8096 13 Version 140530 Human Hsp27 ELISA Kit L ircuLex User s Manual A For Research Use Only Not for use in diagnos
19. tic procedures 2 Precision Intra assay Precision Precision within an assay N Three samples of known concentration were tested 7 times on one plate to assess iN precision sam e Intra assay Within Run n 7 CV 4 4 6 1 Human Hsp27 conc ng mL No Sample 1 Sample 2 Sample 3 1315 546 3 7035 1 1 2 126 4 528 6 8011 4 3 129 1 518 5 7972 8 4 129 6 545 6 5 118 7 488 7 6 113 3 497 2 7 115 1 Inter assay Precision Precision between assays Three samples of known concentration O in three separate assays to assess inter assay precision sample cell lysate e Inter assay Run to Run n 3 CV 1 3 6 7 uman Hsp27 conc ng mL Sample 1 Sample 2 Sample 3 1 129 2 548 0 7347 7 125 5 543 3 8378 5 2 129 9 533 7 7698 7 ax 129 9 548 0 8378 5 min 125 5 533 7 7347 7 Q mean 128 2 541 7 7808 3 SD 2 4 7 3 524 1 CV 1 8 1 3 6 7 C CY 8096 14 Version 140530 P Di Human Hsp27 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Linearity To assess the linearity of the assay samples containing and or spiked with low and hi concentrations of human Hsp27 were serially diluted with the Dilution Buffer to produce samples with values within the dynamic range of the assay sample celllysate Linearity 500 400 F Ve 300 Hsp27 ng ml 0 0 2 0 4 0 6 0 8 1 C CY 8096 15 Version 140530 Human
20. tle containing 20 mL of 1 N H2SO Ready to use C CY 8096 3 Version 140530 Human Hsp27 ELISA Kit CircuLex Users Manual 4 For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips e Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Vortex mixer s of 450 540 nm e read at a single e Plate reader capable of measuring absorbance in 96 well plates at dual wa e Microplate washer optional Manual washing is possible but not preferable i E Dual wavelengths of 450 550 or 450 595 nm can also be used The O b wavelength of 450 nm which will give a somewhat higher reading e Software package facilitating data generation and analysis opti 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality e Disposable paper towels C CY 8096 4 Version 140530 Human Hsp27 ELISA Kit L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Allow all the components to come to room temperature before use e All microplate strips that are not immediately required should be returned to the zip lock pouch Which must be carefully resealed to avoid moisture absorption e Do not use kit components beyond the indicated kit expi
21. uthan C Ehrmann M The HtrA family of proteases implications for protein composition and cell fate Mol Cell 2002 10 443 455 2 Ciocca DR Oesterreich S Chamness GC McGuire WL Fuqua SA Biologicaldand clinical implications of heat shock protein 27 000 Hsp27 a review J Natl Cancer Inst 1993 85 1558570 3 Jakob U Gaestel M Engel K Buchner J Small heat shock proteins are molecular chaperones J Biol Chem 1993 268 1517 20 4 Doshi BM Hightower LE Lee J The role of Hsp27 and actin in the regulati ndof movement in human cancer cells responding to heat shock Cell Stress Chaperones 2009 149445 57 5 Arrigo AP Paul C Ducasse C Manero F Kretz Remy C Virot S et al Small stress proteins novel negative modulators of apoptosis induced independently of reactive oxygen species Prog Mol Subcell Biol 2002 28 185 204 6 Garrido C Brunet M Didelot C Zermati Y Schmitt E Kroemer G Heat shock proteins 27 and 70 anti apoptotic proteins with tumorigenic properties Cell Cycle 2006 5 2592 601 7 Welsh M J and Gaestel M Stress on Life from Molecules to Man edited by Csermely P New York New York Academy of Sciences 1997 p28 35 8 Williams K Chubb C Huberman E Giometti CS Analysis of differential protein expression in normal and neoplastic human breast epithelial c ll lines Electrophoresis 1998 19 333 43 9 Myung JK Afjehi Sadat L Felizardo Cabatic M Slavc I Lubec G Expressional patterns o
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