Human Caspase-9 ELSIA Kit

Transcription

1 Human Caspase-9 ELSIA Kit Cat. No.:DEIA2065 Pkg.Size:96T Intended use The human Caspase-9 ELISA Kit is an enzyme-linked immunosorbent assay for measurement of human Caspase-9. General Description Caspase-9 is an initiator caspase, encoded by the CASP9 gene. CASP9 orthologs have been identified in all mammals for which complete genome data are available. Unique orthologs are also present in lizards, lissamphibians, and teleosts. The aspartic acid specific protease caspase-9 has been linked to the mitochondrial death pathway. It is activated during programmed cell death ( apoptosis ). Induction of stress signaling pathways JNK/SAPK causes release of cytochrome c from mitochondria and activation of apaf-1 ( apoptosome ), which in turn cleaves the pro-enzyme of caspase-9 into the active form. Principle Of The Test An anti-human Caspase-9 coating antibody is adsorbed onto microwells. Human Caspase-9 present in the sample or standard binds to antibodies adsorbed to the microwells. The polyclonal detection antibody ( rabbit ) binds to human Caspase-9 captured by the first antibody. Following incubation unbound detection antibody is removed during a wash step. Anti-rabbit-IgG-HRP is added and binds to the Detection Antibody. Following incubation unbound anti-rabbit-igg-hrp is removed during a wash step, and substrate solution reactive with HRP is added to the wells. A coloured product is formed in proportion to the amount of human Caspase-9 present in the sample or standard. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from 7 human Caspase-9 standard dilutions and human Caspase-9 concentration determined. Reagents And Materials Provided 1. 1 aluminium pouch with a Microwell Plate coated with monoclonal antibody to human Caspase vial ( 100 μl ) anti-human Caspase-9 polyclonal Detection Antibody ( rabbit ) 3. 1 vial ( 10 μl ) Anti-rabbit-IgG-HRP 4. 2 vials human Caspase-9 Standard lyophilized, 200 ng/ml upon reconstitution 5. 1 vial ( 12 ml ) Sample Diluent 6. 1 vial ( 5 ml ) Assay Buffer Concentrate 20x ( PBS with 1% Tween 20 and 10% BSA ) 7. 1 bottle ( 50 ml ) Wash Buffer Concentrate 20x ( PBS with 1% Tween 20 ) 8. 1 bottle ( 15 ml ) Lysis Buffer 10x 9. 1 vial ( 15 ml ) Substrate Solution ( tetramethyl-benzidine ) vial ( 15 ml ) Stop Solution ( 1M Phosphoric acid ) vial ( 0.4 ml ) Blue-Dye vial ( 0.4 ml ) Green-Dye vial ( 0.4 ml ) Red-Dye Adhesive Films Materials Required But Not Supplied 1. 5 ml and 10 ml graduated pipettes 2. 5 µl to 1000 µl adjustable single channel micropipettes with disposable tips

2 3. 50 µl to 300 µl adjustable multichannel micropipette with disposable tips 4. Multichannel micropipette reservoir 5. Beakers, flasks, cylinders necessary for preparation of reagents 6. Device for delivery of wash solution ( multichannel wash bottle or automatic wash system ) 7. Microwell strip reader capable of reading at 450 nm ( 620 nm as optional reference wave length ) 8. Glass-distilled or deionized water 9. Statistical calculator with program to perform regression analysis Storage Store kit reagents between 2 and 8. Immediately after use remaining reagents should be returned to cold storage ( 2 to 8 ). Specimen Collection And Handling Cell lysate, cell culture supernatant and serum were tested with this assay. Other biological samples might be suitable for use in the assay. Remove serum from the clot as soon as possible after clotting. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Samples should be aliquoted and must be stored frozen at -20 to avoid loss of bioactive human Caspase-9. If samples are to be run within 24 hours, they may be stored at 2 to 8. Avoid repeated freeze-thaw cycles. Prior to assay, the frozen sample should be brought to room temperature slowly and mixed gently. Sample Preparation Numerous extraction protocols can be used. The following protocol is provided as an example of a suitable extraction procedure, but should not be construed as necessarily being the method of choice. Users may wish to experiment with extraction procedures that work best in their hands. 1. For suspension cells: Pellet by centrifugation, remove supernatant and proceed to Addition of Lysis Buffer. 2. For attached cells: Remove supernatant from cells, wash cells once with PBS, harvest cells by scraping and gentle centrifugation, aspirate PBS, leaving an intact cell pellet ( at this point the cell pellet can be frozen at -80 and lysed at a later date ) and proceed to Addition of Lysis Buffer. Addition of Lysis Buffer: Resuspend the pellet in Lysis Buffer ( 1x ) ( we recommend a concentration of 5 x106cells/ml. ), incubate 60 minutes at room temperature with gentle shaking and transfer extracts to microcentrifuge tubes and centrifuge at 1000 x g for 15 minutes. Aliquot the cleared lysate to clean microfuge tubes and continue the test procedure ( Alternatively lysates can be stored at -80 and assayed at a later time. Divide lysates into small aliquots to avoid multiple freeze/thaw cycles. ). Reagent Preparation Buffer Concentrates should be brought to room temperature and should be diluted before starting the test procedure. If crystals have formed in the Buffer Concentrates, warm them gently until they have completely dissolved. 1. Wash Buffer: Pour entire contents ( 50 ml ) of the Wash Buffer Concentrate ( 20x ) into a clean 1000 ml graduated cylinder. Bring to final volume of 1000 ml with glass-distilled or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2 to 25. Please note that Wash Buffer ( 1x ) is stable for 30 days. 2. Assay Buffer ( 1x ): Pour the entire contents ( 5 ml ) of the Assay Buffer Concentrate ( 20x ) into a clean 100 ml graduated cylinder. Bring to final volume of 100 ml with distilled water. Mix gently to avoid foaming. Store at 2 to 8. Please note that the Assay Buffer ( 1x ) is stable for 30 days. 3. Lysis Buffer: Pour the entire contents ( 15 ml ) of the Lysis Buffer Concentrate ( 10x ) into a clean 150 ml graduated cylinder. Bring to final volume of 150 ml with distilled or deionized water and mix gently. Store at room temperature. Please note that the Lysis Buffer ( 1x ) is stable for 30 days. 4. Detection Antibody: Please note that the Detection Antibody should be used within 30 minutes after dilution. Make a 1:100

3 dilution of the concentrated Detection Antibody solution with Assay Buffer ( 1x ) in a clean plastic tube. 5. Anti-Rabbit-IgG-HRP: Please note that the anti-rabbit-igg-hrp should be used within 30 minutes after dilution. Make a 1:2000 dilution of the concentrated anti-rabbit-igg-hrp solution with Assay Buffer ( 1x ) in a clean plastic tube. 6. Human Caspase-9 Standard: Reconstitute human Caspase-9 standard by addition of distilled water. Reconstitution volume is stated on the label of the standard vial. Swirl or mix gently to insure complete and homogeneous solubilisation ( concentration of reconstituted standard = 200 ng/ml ). Allow the standard to reconstitute for minutes. Mix well prior to making dilutions. After usage remaining standard cannot be stored and has to be discarded. 7. Addition of Colour-giving Reagents: Blue-Dye, Green-Dye, Red-Dye: In order to help our customers to avoid any mistakes in pipetting the ELISAs, a tool is offered that helps to monitor the addition of even very small volumes of a solution to the reaction well by giving distinctive colours to each step of the ELISA procedure. Assay Steps 1. Prepare cell lysates according to protocol. 2. Determine the number of microwell strips required. 3. Wash microwell strips twice with Wash Buffer. 4. Standard dilution on the microwell plate: Add 100 μl Sample Diluent, in duplicate, to all standard wells. Pipette 100 μl prepared standard into the first wells and create standard dilutions by transferring 100 μl from well to well. Discard 100 μl from the last wells. Alternatively external standard dilution in tubes : Pipette 100 μl of these standard dilutions in the microwell strips. 5. Add 100 μl Sample Diluent, in duplicate, to the blank wells. 6. Add 50 μl Sample Diluent to sample wells. 7. Add 50 μl sample in duplicate, to designated sample wells. 8. Prepare Detection Antibody. 9. Add 50 μl Detection Antibody to all wells. 10. Cover microwell strips and incubate 2 hours at room temperature ( 18 to 25 ). 11. Prepare anti-rabbit-igg-hrp. 12. Empty and wash microwell strips 3 times with Wash Buffer. 13. Add 100 μl diluted anti-rabbit-igg-hrp to all wells. 14. Cover microwell strips and incubate 1 hour at room temperature ( 18 to 25 ). 15. Empty and wash microwell strips 3 times with Wash Buffer. 16. Add 100 μl of TMB Substrate Solution to all wells. 17. Incubate the microwell strips for about 10 minutes at room temperature ( 18 to 25 ). 18. Add 100 μl Stop Solution to all wells. 19. Blank microwell reader and measure colour intensity at 450 nm. Note: If instructions in this protocol have been followed samples have been diluted 1:2 ( 50 μl sample add with 50 μl Sample Diluent ), the concentration read from the standard curve must be multiplied by the dilution factor ( x 2 ). Calculation 1. Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20 per cent of the mean value. 2. Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the human Caspase-9 concentration on the abscissa. Draw a best fit curve through the points of the graph ( a 5-parameter curve fit is recommended ). 3. To determine the concentration of circulating human Caspase-9 for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding human Caspase-9 concentration. 4. If instructions in this protocol have been followed samples have been diluted 1:2 ( 50 µl sample + 50 µl Sample Diluent ), the

4 concentration read from the standard curve must be multiplied by the dilution factor ( x 2 ). 5. Calculation of samples with a concentration exceeding standard 1 may result in incorrect, low human Caspase-9 levels. Such samples require further external predilution according to expected human Caspase-9 values with Sample in order to precisely quantitate the actual human Caspase-9 level. 6. It is suggested that each testing facility establishes a control sample of known human Caspase-9 concentration and runs this additional control with each assay. If the values obtained are not within the expected range of the control, the assay results may be invalid. Typical Standard Curve Representative standard curve for human Caspase-9 ELISA. Human Caspase-9 was diluted in serial 2-fold steps in Sample Diluent. Do not use this standard curve to derive test results. A standard curve must be run for each group of microwell strips assayed. Detection Range ng/ml Sensitivity 0.4 ng/ml Reproducibility

5 % Precautions 1. All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet( s ) and/or safety statement( s ) for specific advice. 2. Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures. 3. Do not mix or substitute reagents with those from other lots or other sources. 4. Do not use kit reagents beyond expiration date on label. 5. Do not expose kit reagents to strong light during storage or incubation. 6. Do not pipette by mouth. 7. Do not eat or smoke in areas where kit reagents or samples are handled. 8. Avoid contact of skin or mucous membranes with kit reagents or specimens. 9. Rubber or disposable latex gloves should be worn while handling kit reagents or specimens. 10. Avoid contact of substrate solutions with oxidizing agents and metal. 11. Avoid splashing or generation of aerosols. 12. In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test use disposable pipette tips and/or pipettes. 13. Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagent. 14. Exposure to acid inactivates the conjugate. 15. Glass-distilled water or deionized water must be used for reagent preparation. 16. Substrate solutions must be at room temperature prior to use. 17. Decontaminate and dispose specimens and all potentially contaminated materials as they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the

6 final mixture contains 1.0% sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite. Limitations 1. Since exact conditions may vary from assay to assay, a standard curve must be established for every run. 2. Bacterial or fungal contamination of either samples or reagents or cross-contamination between reagents may cause erroneous results. 3. Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use. 4. Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh Wash Buffer, fill with Wash Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods. Analyte Gene Information Gene Name CASP9 caspase 9, apoptosis-related cysteine peptidase [ Homo sapiens ] Official Symbol Synonyms CASP9 CASP9; caspase 9, apoptosis-related cysteine peptidase; caspase 9, apoptosis related cysteine protease; caspase-9; APAF 3; ICE LAP6; MCH6; PPP1R56; protein phosphatase 1; regulatory subunit 56; apoptotic protease MCH-6; ICE-like apoptotic protease 6; apoptotic protease activating factor 3; protein phosphatase 1, regulatory subunit 56; APAF3; APAF-3; ICE-LAP6; CASPASE-9c; GeneID 842 mrna Refseq Protein Refseq NM_ NP_ MIM UniProt ID P55211 Chromosome Location 1p36.21 Pathway Function REFERENCES AKT phosphorylates targets in the cytosol, organism-specific biosystem; Activation of caspases through apoptosome-mediated cleavage, organism-specific biosystem; Adaptive Immune System, organism-specific biosystem; Alzheimers disease, organism-specific biosystem; Alzheimers disease, conserved biosystem; Amyotrophic lateral sclerosis (ALS), organism-specific biosystem; Amyotrophic lateral sclerosis (ALS), conserved biosystem; cysteine-type endopeptidase activity; enzyme activator activity; peptidase activity; protein binding; 1. Li P, Nijahawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and datp- dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell Nov 14;91 ( 4 ): Susin SA, Lorenzo HK, Zamzami N, Marzo I, Brenner C, Larochette N, Prevost MC, Alzari PM, Kroemer G. Mitochondrial release of caspase-2 and 9 during the apoptotic process. J Exp Med Jan 18;189( 2 ): Rodriguez J, Lazebnik Y. Caspase-9 and APAF-1 form an active holoenzyme. Genes Dev Dec 15;13( 24 ): Kuida K. Caspase-9. Int J Biochem Cell Biol Feb;32( 2 ):121-4.