Osteopontin ELISA Kit

Produktnummer ABIN7041475

Produktdetails Osteopontin ELISA Kit

Target: Osteopontin (SPP1) (Secreted phosphoprotein 1 (SPP1))

Reaktivität: Hund

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 0.3125 ng/mL - 10 ng/mL

Untere Nachweisgrenze: 0.3125 ng/mL

Applikation: ELISA

Verwendungszweck: The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of osteopontin in dog serum, plasma.

Proben: Plasma, Serum, Urine

Analytische Methode: Quantitative

Bestandteile:

• ELISA Micro Plate antibody coated
• Detection Antibody (100X)
• HRP-Streptavidin (100X)
• Calibrator
• Diluent Solution
• Wash Solution Concentrate (20X)
• Chromogen - Substrate Solution
• STOP Solution

Benötigtes Material:

1. Microplate reader with 450 ± 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Microcentrifuge tubes for diluting samples.
4. Squirt bottle or Microtitre washer
5. Deionized or distilled water.
6. Container for Wash Solution
7. Centrifuge for sample collection
8. Anticoagulant for plasma collection
9. Incubator capable of maintaining 37 °C.
10. Microplate shaker

Anwendungsinformationen

Probenmenge: 100 μL

Testdauer: 3 h

Plattentyp: Pre-coated

Protokoll: In this assay the osteopontin (PONT) present in samples reacts with the anti-PONT antibodies which have been adsorbed to the surface of polystyrene microtitre wells. After the removal of unbound proteins by washing, the Detection Antibody, biotin conjugated anti-PONT, is added and complexes are formed. Following a wash step, the horseradish peroxidase (HRP) conjugated Streptavidin is added and complexes are formed. After another washing step, the complexes are assayed by the addition of a chromogenic substrate, 3,3’,5,5’-tetramethylbenzidine (TMB). The quantity of bound enzyme varies directly with the concentration of PONT in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of PONT in the test sample. The quantity of PONT in the test sample can be interpolated from the standard curve constructed from the standards, and corrected for sample dilution.

Aufbereitung der Reagenzien:

• Bring all reagents to room temperature (16°C to 25°C) before use.
• Diluent Solution - Ready to use as supplied.
• Wash Solution Concentrate - The Wash Solution supplied is a 20X Concentrate and must be diluted 1/20 with distilled or deionized water (1 part buffer concentrate, 19 parts dH2O). Crystal formation in the concentrate may occur when storage temperatures are low. Warming of the concentrate to 30-35°Cbefore dilution can dissolve crystals.
• Detection Antibody - Calculate the required amount of working conjugate solution for each microtitre plate test strip by adding 10 µL Detection Antibody to 990 µL of 1X Diluent for each test strip to be used for testing. Dilute immediately before use and protect from light. Mix uniformly, but gently. Avoid foaming.
• HRP-Streptavidin - Calculate the required amount of working conjugate solution for each microtitre plate test strip by adding 10 µL HRP-Streptavidin to 990 µL of 1X Diluent for each test strip to be used for testing. Dilute immediately before use and protect from light. Mix uniformly, but gently. Avoid foaming.
• Pre-coated ELISA Micro Plate - Ready to use as supplied. Unseal foil pouch and remove plate from pouch. Remove all strips and wells that will not be used in the assay and place back in pouch and re-seal along with desiccant.
• Calibrator – Prepare according to the lot specific Certificate of Analysis.

Aufbereitung der Proben:

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturationmay occur in these samples, leading to false results. Samples should therefore be stored for a short periodat 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thawcycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged toremove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determinecompatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibilityof causing a deviation due to the introduced chemical substance.The recommended dilution factor is for reference only.
• Please estimate the concentration of the samples before performing the test. If the values are not in therange of the standard curve, the optimal sample dilution for the particular experiment has to be determined.Samples should then be diluted with PBS (pH =7.0-7.2).

Note:

The assay requires that each test sample be diluted before use. All samples should be assayed in duplicate each time the assay is performed. The recommended dilutions are only suggestions. Dilutions should be based on the expected concentration of the unknown sample such that the diluted sample falls within the dynamic range of the standard curve. If unsure of sample level, a serial dilution with one or two representative samples before running the entire plate is highly recommended.
Serum samples – Recommended starting dilution is 1/20. To prepare a 1/20 dilution of a sample, transfer 20 µL of sample to 380 µL of 1X diluent. This gives you a 1/20 dilution. Mix thoroughly.
Plasma samples – Recommended starting dilution is 1/20. To prepare a 1/20 dilution of a sample, transfer 20 µL of sample to 380 µL of 1X diluent. This gives you a 1/20 dilution. Mix thoroughly.
Urine samples – Recommended starting dilution is 1/20. To prepare a 1/20 dilution of a sample, transfer 20 µL of sample to 380 µL of 1X diluent. This gives you a 1/20 dilution. Mix thoroughly.

Testdurchführung:

1. All samples and standards should be assayed in duplicates.
   2. The Standards and the test sample(s) should be loaded into the ELISA wells as quickly as possible to avoid a shift in OD readings. Using a multichannel pipette would reduce this occurrence. Pipette 100 μL of Standard 0 (0.0 ng/mL) in duplicate Standard 1 (0.16 ng/mL) in duplicate Standard 2 (0.31 ng/mL) in duplicate Standard 3 (0.63 ng/mL) in duplicate Standard 4 (1.25 ng/mL) in duplicate Standard 5 (2.50 ng/mL) in duplicate Standard 6 (5 ng/mL) in duplicate Standard 7 (10 ng/mL) in duplicate
   3. Pipette 100 μL of sample (in duplicate) into pre designated wells.
   4. Incubate the micro titer plate while shaking at 400rpm on a microplate shaker at room temperature for one hundred and twenty (120 ± 2) minutes. Keep plate covered and level during incubation.
   5. Following incubation, aspirate the contents of the wells.
   6. Completely fill each well with appropriately diluted Wash Solution and aspirate. Repeat three times, for a total of four washes. If washing manually: completely fill wells with wash buffer, invert the plate then pour/shake out the contents in a waste container. Follow this by sharply striking the wells on absorbent paper to remove residual buffer. Repeat 3 times for a total of four washes.
   7. Pipette 100 μL of appropriately diluted Detection Antibody to each well. Incubate while shaking at 400rpm on a microplate shaker at room temperature for twenty (20 ± 2) minutes. Keep plate covered in the dark and level during incubation.
   8. Wash and blot the wells as described in Steps 5/6.
   9. Pipette 100 μL of appropriately diluted HRP-streptavidin to each well. Incubate while shaking at 400rpm on a microplate shaker at room temperature for twenty (20 ± 2) minutes. Keep plate covered in the dark and level during incubation.
   10. Wash and blot the wells as described in Steps 5/6.
   11. Pipette 100 μL of TMB Substrate Solution into each well.
   12. Incubate while shaking at 400rpm on a microplate shaker in the dark at room temperature for precisely ten (10) minutes.
   13. After ten minutes, add 100 μL of Stop Solution to each well.
   14. Determine the absorbance (450 nm) of the contents of each well within 30 minutes. Calibrate the plate reader to manufacturer’s specifications.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Informationen zur Lagerung:

1. For unopened kit: All reagents should be stored according to the labels on the vials. All reagents should be stored at 4 °C for long term storage. Keep away from heat or direct sunlight.
2. For opened kits: the remaining reagents must be stored according to the above storage conditions. In addition, please return the unused wells to the foil pouch containing the desiccant and seal the foil pouch with the zipper.

.

Haltbarkeit: 6 months

Details zu Osteopontin

Target: Osteopontin (SPP1) (Secreted phosphoprotein 1 (SPP1))

Andere Bezeichnung: Osteopontin (SPP1 Produkte)

Synonyme: BNSP ELISA Kit, BSPI ELISA Kit, ETA-1 ELISA Kit, OPN ELISA Kit, 2AR ELISA Kit, Apl-1 ELISA Kit, Bsp ELISA Kit, Eta ELISA Kit, OP ELISA Kit, Opn ELISA Kit, Opnl ELISA Kit, Ric ELISA Kit, Spp-1 ELISA Kit, OSP ELISA Kit, zgc:111821 ELISA Kit, secreted phosphoprotein 1 ELISA Kit, SPP1 ELISA Kit, Spp1 ELISA Kit, spp1 ELISA Kit

Pathways: Regulation of Cell Size