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CBA048 Survivin ELISA Kit

Survivin ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
CBA048
  
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Tabulka spec. kláve

Detection Methods
Colorimetric
Description
Overview

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Survivin, a member of the inhibitor of apoptosis proteins (IAPs) family, is highly expressed in a wide range of tumor cells, but is not expressed in the majority of normal differentiated adult tissue. This ELISA detects and quantifies native and recombinant human survivin in cell lysates.
Catalogue NumberCBA048
Brand Family Calbiochem®
Application Data
A standard curve should be generated for each set of samples assayed. The graph below represents typical data generated when using the Survivin ELISA Kit. The standard curve was calculated using a computer generated 4-PL curve-fit. This standard curve is for demonstration purposes only.
Materials Required but Not Delivered• Aprotinin (Cat No. 616398)
• Leupeptin (Cat. No. 108975)
• Pepstatin (Cat. No. 516481)
• Phenylmethylsulfonylfluoride (PMSF) (Cat No. 52332)
• Urea
• Triton® X-100-detergent
• Pipettes and pipette tips
• Deionized or distilled water
• 96 well plates [Costar EIA Plates is recommended]
• Plate sealers
• Squirt bottle, manifold dispenser, or automated plate washer
• PBS-137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2-7.4, 0.2 µm filtered.
• Wash Buffer - 0.05% Tween®- 20 detergent in PBS, pH 7.2-7.4.
• Block Buffer - 1% BSA, 0.05% NaN3, in PBS, pH 7.2-7.4.
• IC Diluent #1 - 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered.
• IC Diluent #2 - 1% BSA, 1 M urea in PBS, pH 7.2-7.4, 0.2 µm filtered.
Note: Approximately 50 ml of this diluent is required to run the assay on one plate.
• IC Diluent #5 - 1 mM EDTA, 0.5% Triton® X-100-detergent, 6 M urea in PBS, pH 7.2-7.4.
• Lysis Buffer #2 - 1 mM EDTA, 0.5% Triton® X-100-detergent, 6 M urea, 10 µg/ml Leupeptin, 10 µg/ml Pepstatin, 100 µM PMSF, 3 µg/ml Aprotinin in PBS, pH 7.2-7.4.
• Substrate Solution - 1:1 mixture of H2O2 and Tetramethylbenzidine
• Stop Solution - 2 N H2SO4
References
Product Information
Detection methodColorimetric
Form182 Tests
Format96-well plate
Kit containsTotal Survivin Capture Antibody, Total Survivin Detection Antibody, Total Survivin Standard, Streptavidin-HRP, and a user protocol.
Applications
Biological Information
Assay time9-10 h
Sample TypeCells
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Standard Handling
Storage +2°C to +8°C
Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit containsTotal Survivin Capture Antibody, Total Survivin Detection Antibody, Total Survivin Standard, Streptavidin-HRP, and a user protocol.
Specifications
Global Trade Item Number
Katalogové číslo GTIN
CBA048 0

Documentation

Survivin ELISA Kit Certificates of Analysis

TitleLot Number
CBA048
User Protocol

Revision20-October-2008 RFH
Form182 Tests
Format96-well plate
Detection methodColorimetric
Specieshuman
StorageUpon arrival store the entire contents of the kit at 4°C.
Principles of the assayThe Calbiochem® Survivin ELISA Kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human survivin in cell lysates. An immobilized capture antibody binds survivin present in samples or standards. After washing away unbound material, a biotinylated detection antibody specific for survivin is used to detect the protein, utilizing a standard streptavidin-HRP format.
Materials provided• Total Survivin Capture Antibody (Kit Component No. JA9148-1EA): 1 vial
• Total Survivin Detection Antibody (Kit Component No. JA9149-1EA): 1 vial
• Total Survivin Standard (Kit Component No. JA9150-1EA): 3 vials
• Streptavidin-HRP (Kit Component No. JA9151-1EA): 1 vial

The Survivin ELISA Kit contains sufficient materials to run ELISAs on at least two 96 well plates provide the reagents are prepared as described in this User Protcol, the assay is run as described in the Detailed Protocol, and the recommended plates, buffers, diluents, substrates, and solutions are used.
Materials Required but not provided• Aprotinin (Cat No. 616398)
• Leupeptin (Cat. No. 108975)
• Pepstatin (Cat. No. 516481)
• Phenylmethylsulfonylfluoride (PMSF) (Cat No. 52332)
• Urea
• Triton® X-100-detergent
• Pipettes and pipette tips
• Deionized or distilled water
• 96 well plates [Costar EIA Plates is recommended]
• Plate sealers
• Squirt bottle, manifold dispenser, or automated plate washer
• PBS-137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2-7.4, 0.2 µm filtered.
• Wash Buffer - 0.05% Tween®- 20 detergent in PBS, pH 7.2-7.4.
• Block Buffer - 1% BSA, 0.05% NaN3, in PBS, pH 7.2-7.4.
• IC Diluent #1 - 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered.
• IC Diluent #2 - 1% BSA, 1 M urea in PBS, pH 7.2-7.4, 0.2 µm filtered.
Note: Approximately 50 ml of this diluent is required to run the assay on one plate.
• IC Diluent #5 - 1 mM EDTA, 0.5% Triton® X-100-detergent, 6 M urea in PBS, pH 7.2-7.4.
• Lysis Buffer #2 - 1 mM EDTA, 0.5% Triton® X-100-detergent, 6 M urea, 10 µg/ml Leupeptin, 10 µg/ml Pepstatin, 100 µM PMSF, 3 µg/ml Aprotinin in PBS, pH 7.2-7.4.
• Substrate Solution - 1:1 mixture of H2O2 and Tetramethylbenzidine
• Stop Solution - 2 N H2SO4
Precautions and recommendations• The Stop Solution suggested for use with this kit is an acidic solution. Wear eye, hand, face, and clothing protection when using this material.
• Individual results may vary due to differences in technique, plasticware and water sources.
• It is important that the diluents selected for reconstitution and for dilution of the standard reflect the environment of the samples being measured. The diluents suggested in this protocol should be suitable for most cell lysates.
• The type of enzyme and substrate and the concentrations of capture/detection antibodies used can be varied to create an immunoassay with a different sensitivity and dynamic range. A basic understanding of immunoassay development is required for the successful use of these reagents in immunoassays.
• A thorough and consistent wash technique is essential for proper assay performance. Wash Buffer should be dispensed forcefully and removed completely from the wells by aspiration or decanting. Remove any remaining Wash Buffer by inverting the plate and blotting it against clean paper towels.
• Use a fresh reagent reservoir and pipette tips for each step.
• It is recommended that all standards and samples be assayed in duplicate.
• Avoid microbial contamination of reagents and buffers. This may interfere with the sensitivity of the assay. Buffers containing protein should be made under sterile conditions and stored at 4°C or be prepared fresh daily.
Preparation• Preparation of Cell Lysates: 1. Rinse cells two times with PBS, making sure to remove any remaining PBS after the second rinse. 2. Solubilize cells at 1 x 107 cells/ml in Lysis Buffer #2. 3. Vortex lysates briefly and allow to sit on ice for 15 min or store at -20°C in a manual defrost freezer. 4. Sample protein concentration may be quantified using a total protein assay. 5. Before use, centrifuge at 2000 x g for 5 min and transfer the supernatant into a clean test tube. 6. Dilute lysates 6-fold with IC Diluent #1 and make further serial dilutions in IC Diluent #2. Note: The final concentration of urea in all samples and standards should be 1 M prior to addition to the plate.
Reagent preparationBring all reagents to room temperature before use. • Total Survivin Capture Antibody: 72 µg/ml rabbit anti-human survivin antibody when reconstituted with 200 µl PBS. After reconstitution, store at 4°C for up to 30 days or aliquot and store at -20°C in a manual defrost freezer or at -70°C for up to 3 months. • Total Survivin Detection Antibody: 7.2 µg/ml biotinylated rabbit anti-human survivin antibody when reconstituted with 1 ml IC Diluent #1. After reconstitution, store at 4°C for up to 30 days or aliquot and store at -20°C in a manual defrost freezer or at -70°C for up to 3 months. • Total Survivin Standard: 180 ng/ml recombinant human Survivin when reconstituted with 500 µl IC Diluent #5. After reconstitution, aliquot and store the Standard at 4°C for up to 30 days or at -70°C for up to 1 month. Immediately before use, an initial 6-fold dilution should be made in IC Diluent #1. Further dilutions should be made in IC Diluent #2 immediately before use. A seven point standard curve using 2-fold serial dilutions and a high standard of 4000 pg/ml is recommended. • Streptavidin-HRP: 1 ml Streptavidin conjugated to horseradish-peroxidase. Store at 4°C. DO NOT FREEZE.
Detailed protocol1. Dilute the Total Survivin Capture Antibody to the working concentration of 0.4 µg/ml in PBS without carrier protein. Immediately coat a 96-well plate with 100 µl per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of 3 washes. Wash by filling each well with Wash Buffer (400 µl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
3. Block plates by adding 300 µl Block Buffer to each well. Incubate at room temperature for 1-2 h.
4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
5. Add 100 µl sample or standards in IC Diluent #2 per well. Use IC Diluent #2 as the zero standard. Cover with a plate sealer and incubate 2 h at room temperature.
Note: A seven point standard curve using 2-fold serial dilutions and a high standard of 4000 pg/ml is recommended.
6. Repeat the aspiration/wash as in step 2.
7. Dilute the Total Survivin Detection Antibody to a working concentration of 200 ng/ml in IC Diluent #1 immediately before use. Add 100 µl diluted Total Survivin Detection Antibody to each well. Cover with a new plate sealer and incubate 2 h at room temperature.
8. Repeat the aspiration/wash as in step 2.
9. Immediately before use, dilute the Streptavidin-HRP to the working concentration specified on the vial label using IC Diluent #1. Add 100 µl diluted Streptavidin-HRP to each well. Incubate for 20 min at room temperature. Avoid placing the plate in direct light.
10. Repeat the aspiration/wash as in step 2.
11. Add 100 µl Substrate Solution to each well. Incubate for 20 min at room temperature. Avoid placing the plate in direct light.
12. Add 50 µl Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
13. Determine the absorbance of each well immediately, using a plate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
14. Calculation of results
a. Average the duplicate readings for each standard and sample, then subtract the average zero standard absorbance. Results may be normalized to total protein or cell number.
b. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the survivin concentrations versus the log of the absorbance and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data.
Standard curveThe Survivin ELISA Kit is calibrated against a highly purified E. coli-expressed recombinant human survivin.

Figure 1: Typical Standard Curve

A standard curve should be generated for each set of samples assayed. The graph below represents typical data generated when using the Survivin ELISA Kit. The standard curve was calculated using a computer generated 4-PL curve-fit. This standard curve is for demonstration purposes only.
SpecificityThe Survivin ELISA Kit is specific for survivin. Specificity was demonstrated by immunoblot analysis of the protein bound by the capture antibody supplied in the kit (see Figure 1).

Figure 2: Specificity

Lysates prepared from human HepG2 cells, either untreated (-) or treated (+) with the G2/M blocking agent nocodazole, were incubated in wells coated with the Total Survivin Capture Antibody. Unbound material was removed by washing and bound material was solubilized in SDS gel sample buffer. Captured proteins were electrophoresed, transferred to an Immobilon-P (Millipore) membrane and immunoblotted with the Total Survivin Detection Antibody. Only a single band corresponding to human survivin was detected.


Quantitation

Absolute amounts of human survivin, as measured by the Survivin ELISA Kit, are consistent with the amounts of human survivin determined by qualitative immunoblot analysis (see Figure 2).

Figure 3: Quantitation of human Survivin in THP-1 and HeLa cells

THP-1 and HeLa cells were either untreated (-) or treated (+) with 200 ng/ml nocodazole for 16 h. Cells were lysed as described in the Preparation of Samples section. Human survivin was measured with the Survivin ELISA Kit. The same lysates were immunoblotted (inset) with rabbit anti-human survivin. The ELISA results correlate well with survivin amounts detected by immunoblot.
Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
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Tween® is a registered trademark of ICI Americas, Inc.
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