QIA23 Bcl-2 ELISA Kit

QIA23
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      Overview

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      Key Specifications Table

      Species ReactivityDetection Methods
      H Colorimetric

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      QIA23-1EA
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          This product has been discontinued.



          A quantitative colorimetric assay for the measurement of Bcl-2. Able to detect significant decreases in Bcl-2 protein levels before significant levels of apoptosis are detected. Kit has high sensitivity, a broad dynamic range, and contains all required buffers.

          One unit of Bcl-2 equals the Bcl-2 activity in 5.6 X 104 HL-60 cells
          Catalogue NumberQIA23
          Brand Family Calbiochem®
          Application Data
          Neutralization of bcl-2 positive samples using a bcl-2 monoclonal antibody (Cat. No. OP60).

          The pooled coefficient of variation (according to the formula of Henry, et al., 1974) is plotted against bcl-2 concentrations. The pooled data were collected from samples run (in replicates of four) on two separate occasions using two different lots of coated plates on each occasion (total of four assays). The triangles represent samples run on one occasion.

          The pooled coefficient of variation (according to the formula of Henry, et al., 1975) is plotted against bcl-2 concentrations. The pooled data were collected from samples run (in replicates of four) on two separate occasions using two different lots of coated plates on each occasion (total of four assays). The triangles represent samples run on one occasion.

          Human sera and plasma samples were spiked with bcl-2 protein to obtain a final concentration of 42 units/ml and analyzed as outlined above. The control represents bcl-2 spiked into sample diluent.

          Dilution recovery from a bcl-2 positive cell line using different antigen extraction agents (different detergents).

          Dilution recovery using different cell lines.
          Materials Required but Not Delivered 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips
          Wash bottle or multichannel dispenser for washing.
          500 ml graduated cylinder.
          Deionized or distilled H2O.
          Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/540 nm or 450/595. A single wavelength of 450 nm can also be used.
          Cell Resuspension Buffer: 50 mM Tris, containing 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin adjusted to pH 7.4
          References
          ReferencesKorsmeyer, S.J., 1995. Trends Genet. 11, 101.
          Hockenbery D.M., 1995. BioEssays 17, 631.
          Steinman, H.M., 1995. J. Biol. Chem. 270, 3487.
          Hawkins, C.J., and Vaux, D., 1994. Immunol. Rev. 142, 127.
          Hengartner, M.O., et al. 1994. Nature 369, 318.
          Ellis, R.E., et al. 1994. Soc. London Ser. 34, 243.
          Zoltan, N., et al. 1994. Cell 79, 189.
          Tsujimoto, Y. 1989. Oncogene 4, 1331.
          Product Information
          Detection methodColorimetric
          Form96 Tests
          Format96-well plate
          Kit contains96 Well Coated Plate, Antigen Extraction Reagent, Lyophilized Bcl-2 Standard, Sample Diluent, Anti-FITC Conjugated Detector Antibody, Concentrated Anti-FITC Peroxidase Conjugate, Conjugate Diluent, Substrate, Wash Buffer, Stop Solution, Adhesive Plate Sealers, and a user protocol.
          Applications
          Key Applications Enzyme-Linked Immunosorbent Assay
          Biological Information
          Assay time3.5 h
          Sample TypeCell lysates, tissue culture supernatant, sera, or plasma
          Species Reactivity
          • Human
          Physicochemical Information
          Sensitivity< 1 ng/ml
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 23/24/25-35-36/37/38-40-43

          Toxic by inhalation, in contact with skin and if swallowed.
          Causes severe burns.
          Irritating to eyes, respiratory system and skin.
          Limited evidence of a carcinogenic effect.
          May cause sensitization by skin contact.
          S PhraseS: 26-36/37/39-45

          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Wear suitable protective clothing, gloves and eye/face protection.
          In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
          Product Usage Statements
          Intended useThe Calbiochem® bcl-2 ELISA is a non-isotopic immunoassay for the in vitro quantitation of human bcl-2 protein in cell extracts, tissue culture medium, sera and plasma. This assay is not recommended for use with rodent samples.
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
          Storage -20°C
          Storage ConditionsUpon receipt, standards must be stored at -20°C. All other components may be stored at -20°C or 4°C (do not refreeze after thawing). Do not expose reagents to excessive light. Allow kit reagents to warm to room temperature before use.
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit contains96 Well Coated Plate, Antigen Extraction Reagent, Lyophilized Bcl-2 Standard, Sample Diluent, Anti-FITC Conjugated Detector Antibody, Concentrated Anti-FITC Peroxidase Conjugate, Conjugate Diluent, Substrate, Wash Buffer, Stop Solution, Adhesive Plate Sealers, and a user protocol.
          Specifications

          Documentation

          Bcl-2 ELISA Kit SDS

          Title

          Safety Data Sheet (SDS) 

          Bcl-2 ELISA Kit Certificates of Analysis

          TitleLot Number
          QIA23

          References

          Reference overview
          Korsmeyer, S.J., 1995. Trends Genet. 11, 101.
          Hockenbery D.M., 1995. BioEssays 17, 631.
          Steinman, H.M., 1995. J. Biol. Chem. 270, 3487.
          Hawkins, C.J., and Vaux, D., 1994. Immunol. Rev. 142, 127.
          Hengartner, M.O., et al. 1994. Nature 369, 318.
          Ellis, R.E., et al. 1994. Soc. London Ser. 34, 243.
          Zoltan, N., et al. 1994. Cell 79, 189.
          Tsujimoto, Y. 1989. Oncogene 4, 1331.
          User Protocol

          Revision22-July-2008 RFH
          Form96 Tests
          Format96-well plate
          Detection methodColorimetric
          Specieshuman
          StorageUpon receipt, standards must be stored at -20°C. All other components may be stored at -20°C or 4°C (do not refreeze after thawing). Do not expose reagents to excessive light. Allow kit reagents to warm to room temperature before use.
          Intended useThe Calbiochem® bcl-2 ELISA is a non-isotopic immunoassay for the in vitro quantitation of human bcl-2 protein in cell extracts, tissue culture medium, sera and plasma. This assay is not recommended for use with rodent samples.
          BackgroundExpression of bcl-2 promotes cell survival by countering some but not all stimuli known to induce cell death. The bcl-2 proto-oncogene is encoded by a gene 230 kb in size that gives rise to a 24-26 kDa protein which has been localized to the mitochondrial, microsomal and nuclear membrane sites within many cell types. The mammalian gene bcl-2 is homologous to the ced-9 gene in the nematode worm Caenorhadbitis elegans (C. elegans). The proteins are 23% identical in sequence and human bcl-2 can function in C. elegans ced-9 negative cells to suppress apoptosis, indicating that the control of apoptosis has been highly conserved during evolution. Although data exists for implicating bcl-2 as a novel GTP-binding protein, the major function of bcl-2 appears to be an inhibitor of apoptosis by an as yet undefined mechanism. It is well documented that bcl-2 becomes deregulated in tumor cells as a result of translocation into the immunoglobulin heavy-chain locus and is therefore constitutively activated in B cell malignancies (i.e. follicular lymphoma and chronic lymphocytic leukemia). In addition, bcl-2 has also been shown to protect mammalian cells from DNA, RNA, and protein-synthesis inhibitors, as well as from sodium azide, colchicine, steroids, heat shock and irradiation treatments. bcl-2 can inhibit apoptosis caused by a variety of physiological and pathological stimuli. Although it has been shown that the phosphorylation state of bcl-2 is critical to its ability to inhibit apoptosis, the exact mechanism of action through which bcl-2 exerts its effects is largely unknown. One intriguing observation is that bcl-2 may function as a pro-oxidant regulating levels of reactive oxygen intermediates and controlling early entry into apoptosis.
          Principles of the assayThe bcl-2 ELISA is a "sandwich" enzyme immunoassay employing mouse monoclonal antibodies. An antibody, specific for the human bcl-2 protein, has been immobilized onto the surface of the plastic wells provided in the kit. The sample to be assayed and FITC conjugated detector monoclonal antibody are pipetted into the wells and allowed to incubate for 2 h, during which any bcl-2 present binds to the capture and detecting antibodies. Unbound material is washed away and horseradish peroxidase-conjugated anti-FITC antibody is added, which binds to the detector antibody. The horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of bcl-2 protein in the sample. The colored reaction product is quantified using a spectrophotometer.
          Quantitation is achieved by the construction of a standard curve using known concentrations of bcl-2 (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of bcl-2 with that obtained from the standards, the concentration of bcl-2 in the sample can be determined.
          Materials providedStandards should be assayed in duplicate. A standard curve must be performed on the same plate and at the same time as the samples. The bcl-2 ELISA provides sufficient reagents to run two sets of standard curves, and 42 samples (if assayed in duplicate all at once using one standard curve), or 36 samples (if assayed on two separate occasions using two standard curves).

          • COATED 96-WELL PLATE (Kit Component No. JA1518): 96 removable wells coated with bcl-2 monoclonal antibody.
          • bcl-2 STANDARD (Kit Component No. JA1519): Two (2) vials containing lyophilized bcl-2 recombinant protein. Reconstituted standards should be discarded after one use. Note: the standard was previously supplied as a lysate and reported in units/ml; it is now being supplied as a recombinant protein and reported in ng/ml
          • DETECTOR ANTIBODY (Kit Component No. JA1520): One (1) bottle containing a 7 ml solution of FITC-Conjugated monoclonal anti-human bcl-2 antibody.
          • 200X CONJUGATE (Kit Component No. JA1521): Rabbit anti-FITC Peroxidase Conjugate: 200-fold concentrated solution (0.1 ml).
          • CONJUGATE DILUENT (Kit Component No. JA1522): 12 ml of the buffer for dilution of 200X Conjugate.
          • AEA (Antigen Extraction Agent) (Kit Component No. JA1523): Two (2) vials containing 3 ml each of AEA. Use as directed in sample preparation section for the extraction of bcl-2 from cell and tissue preparations.
          • SUBSTRATE (Kit Component No. JA1608): 12 ml of the chromogenic substrate.
          • SAMPLE DILUENT (Kit Component No. JA1525): 25 ml of buffer used to dilute standards and samples.
          • 20X PLATE WASH CONCENTRATE (Kit Component No. JA1617): 100 ml of 20-fold concentrated solution of PBS and surfactant. Contains 2% chloroacetamide.
          • STOP SOLUTION (Kit Component No. JA1616): 2.5 N sulfuric acid (12 ml)
          • PLATE SEALERS (Kit Component No. JB155): To cover plates during incubations.
          Materials Required but not provided 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips
          Wash bottle or multichannel dispenser for washing.
          500 ml graduated cylinder.
          Deionized or distilled H2O.
          Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/540 nm or 450/595. A single wavelength of 450 nm can also be used.
          Cell Resuspension Buffer: 50 mM Tris, containing 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin adjusted to pH 7.4
          Precautions and recommendations Store standards at -20°C. All other components may be stored at -20°C or 4°C (do not refreeze after thawing). Do not expose reagents to excessive light. Warm kit reagents to room temperature before use (let sit at room temperature approximately 30 min before use.)
          Wear disposable gloves and eye protection.
          Use only the wells provided with the kit.
          Do not mix reagents from different kits.
          Do not mouth pipette or ingest any of the reagents.
          The buffers and reagents used in this kit contain anti-microbial and anti-fungal reagents. Care should be taken to prevent direct contact with these products.
          Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled.
          Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
          PreparationCell Lysates: Numerous extraction protocols can be used. The following protocol has been shown to work with a number of cell lines. It 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 step # 3. For attached cells, remove supernatant from cells (you may test the supernatant by adding 0.2 ml AEA per 1 ml of supernatant). 2. Wash cells once with PBS, harvest cells by scraping and gentle centrifugation. 3. Aspirate PBS leaving an intact cell pellet (at this point the cell pellet can be frozen at -80°C and lysed at a later date). We recommend for every 5 X 106 cells, resuspend the pellet in 1 ml of Resuspension Buffer (50 mM Tris, containing 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin adjusted to pH 7.4). 4. Add 20 µl of Antigen Extraction Agent (AEA provided) for every 100 µl of cell suspension. 5. Incubate 30 min on ice with occasional vortexing. 6. Transfer extracts to microcentrifuge tubes and centrifuge for 5 min. 7. Aliquot cleared lysate to clean microfuge tubes. These samples are now ready for analysis according to the instructions provided in Detailed Protocol. Lysates can be frozen at -80°C and assayed at a later time. The sample should be divided into small aliquots to avoid multiple freeze/thaw cycles. Note: Samples found to contain greater than 200 units/ml bcl-2 (i.e., outside the range of the standard curve) must be diluted with Sample Diluent (provided), so that the bcl-2 concentration falls within the range spanned by the standard curve, and assayed again. Sera and Plasma: Sera and plasma samples can be prepared using standard protocols and analyzed according to the instructions provided in Detailed Protocol. Samples can be frozen at -80°C and assayed at a later time. The sample should be divided into small aliquots to avoid multiple freeze/thaw cycles. Tissue Culture Media: For suspension cells. Pellet by centrifugation (1000 x g for 10 min, 4°C) and remove supernatant for testing. Samples may be stored at -20°C. For adherent cells. Remove tissue culture media, centrifuge tissue culture media (1000 x g for 10 min), and remove supernatant for testing. Samples may be store at -20°C.
          Detailed protocolThe bcl-2 ELISA is provided with removable strips of wells so the assay can be carried out on two separate occasions. Since conditions may vary, a standard curve MUST be determined each time the assay is performed. Standards should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples.

          1. Remove the appropriate number of wells from the foil pouch and place them into the empty well holder. Return any unused wells to the foil pouch, refold, seal with tape and store at 4°C. Let all other kit components sit at room temperature until used. Best results will be obtained using reagents at room temperature.
          2. Prepare a working solution (1X) of Wash Buffer by adding 25 ml of the 20X concentrated solution (provided), to 475 ml of deionized water. Mix well.
          3. Each time an assay is performed, reconstitute a Lyophilized Standard as described on the lyophilized bcl-2 STANDARD vial label to give a concentration of 100 ng/ml. Let the reconstituted standard sit for 15 min, with occasional swirling. Avoid excessive agitation of the standard. After reconstituting the bcl-2 Lyophilized Standard it should be diluted with Sample Diluent. Obtain seven tubes and label them 100, 50, 25, 12.5, 6.25, 3.25, and 0 ng/ml. Add 300 µl of Sample Diluent into each tube except the 100 ng/ml tube (first tube) which gets "undiluted" reconstituted standard. Remove 600 µl from the original vial of lyophilized material and add it to the first tube. Remove 300 µl from the first tube (100 ng/ml) and add it to the second tube (50 ng/ml) and mix gently. Repeat this procedure until you reach the fifth tube (3.25 ng/ml). The last tube (0 ng/ml) should just be Sample Diluent. Reconstituted standards should be discarded after one use.
          4. Prepare all samples. Cell lysates should be diluted with Sample Diluent 1:5 or greater as needed, sera and plasma samples as well as tissue culture media samples should be diluted with Sample Diluent 1:10 or greater as needed.
          5. Pipette 50 µl of the Detector Antibody into each well.
          6. Add samples and each of the bcl-2 standards (in duplicate) by pipetting 50 µl into appropriate wells using clean pipette tips for each sample.
          7. Cover wells with a plate sealer and incubate at room temperature for 2 h.
          8. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
          9. Dilute a sufficient amount of the 200X Conjugate 1:200 in Conjugate Diluent to provide 100 µl of 1X solution for each of the sample and standard wells, mix gently. For example: add 60 µl to 12 ml of Conjugate Diluent. Filter with a 0.2 µm syringe filter prior to use.
          10. Pipette 100 µl of the Conjugate into each well, cover with a plate sealer and incubate at room temperature for 30 min. Discard any unused 1X Conjugate.
          11. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
          12. FLOOD ENTIRE PLATE WITH dH2O. Remove contents of wells by inverting over sink and tapping on paper towels.
          13. Add 100 µl of Substrate Solution to each well and incubate IN THE DARK at room temperature for 30 min.
          14. Add 100 µl of Stop Solution to each well in the same order as the previously added Substrate Solution.
          15. Measure absorbance in each well using a spectrophotometric plate reader. It is preferable to read at dual wavelengths of 450/595 nm (or 450/540 nm). A single wavelength of 450 nm can also be used. Wells must be read within 30 min of adding the Stop Solution.
          Calculations1. Average the duplicate absorbance values for each standard, including the zero, and all sample values. 2. On graph paper, plot the mean absorbance values for each of the standards on the Y axis, versus the concentration of each standard (units/ml) on the X axis. 3. Determine the concentration of unknowns by interpolation from the standard curve. There are a variety of plate reader software packages available (Softmax, Molecular Devices Corporation, Menlo Park, CA; KinetiCalc, BioTek Instruments, Inc. Winooski, VT) for analysis of plate data, which simplifiesx this process. 4. For samples which have been diluted, the bcl-2 concentration must be multiplied by the dilution factor (ie., if the sample was diluted five fold, then the bcl-2 value obtained from the standard curve must be multiplied by five).

          Figure 1: bcl-2 ELISA: Sample Standard Curve

          The mean signal of of each standard run in replicates of two in four assays using two different lots of plates. Note: 100 units/ml of the old standard correspond to ~10 ng/ml of the new standard.

          Assay characteristics and examplesThe bcl-2 ELISA can detect bcl-2 protein in human sera and plasma samples and cell lysates. Specificity was demonstrated by neutralization (inhibition of assay signal) of bcl-2 positive human sera samples and cell lysates by a specific bcl-2 monoclonal antibody. The neutralizing bcl-2 antibody, which is not a component of the ELISA, inhibited the signal of all positive samples (both cell lysates and sera), while the control antibody (non bcl-2 antibody) did not effect the signal of the bcl-2 positive samples. In addition, samples (cell lysates and sera) that had high levels of bcl-2 protein, as determined by the ELISA, gave a 26 kDa band when analyzed by the bcl-2 Western blot technique.

          Figure 2: Neutralization Assay

          Neutralization of bcl-2 positive samples using a bcl-2 monoclonal antibody (Cat. No. OP60).

          Sensitivity< 1 ng/ml
          Sensitivity NotesThe bcl-2 ELISA can distinguish <1 ng/ml of bcl-2 from zero.
          Precision

          Figure 3: Precision

          The pooled coefficient of variation (according to the formula of Henry, et al., 1974) is plotted against bcl-2 concentrations. The pooled data were collected from samples run (in replicates of four) on two separate occasions using two different lots of coated plates on each occasion (total of four assays). The triangles represent samples run on one occasion.


          Figure 4: Precision

          The pooled coefficient of variation (according to the formula of Henry, et al., 1975) is plotted against bcl-2 concentrations. The pooled data were collected from samples run (in replicates of four) on two separate occasions using two different lots of coated plates on each occasion (total of four assays). The triangles represent samples run on one occasion.

          Recoverybcl-2 spike and recovery in human sera and plasma is close to 100% if the samples are treated with AEA

          Figure 5: Recovery in Human Sera and Plasma

          Human sera and plasma samples were spiked with bcl-2 protein to obtain a final concentration of 42 units/ml and analyzed as outlined above. The control represents bcl-2 spiked into sample diluent.

          LinearityLinearity of Dilution was determined in two separate studies. The first study tested dilution-recovery of one cell line (HL60) extracted with six different antigen extraction agents and the second study tested dilution-recovery of six positive samples using one AEA. The dilutions were run in the bcl-2 ELISA and the "found" doses were plotted against the "expected" doses to determine linearity of dilution. In both studies the slope is not significantly different from one and the intercept is very close to zero. These studies are consistent with the absence of cross-reacting and matrix effects such as pH, salts, and endogenous binders that interfere with the reagents used in the assay.

          Figure 6: Dilution Recovery With Different AEA

          Dilution recovery from a bcl-2 positive cell line using different antigen extraction agents (different detergents).

          Figure 7: Dilution Recovery With Different Cell Lines

          Dilution recovery using different cell lines.

          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.