BMP4 ELISA Kit

Catalog No. ABIN624951

Product Details BMP4 ELISA Kit

Target: BMP4 (Bone Morphogenetic Protein 4 (BMP4))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: Human BMP-4 ELISA Kit for cell and tissue lysate samples.

Sample Type: Cell Lysate, Tissue Lysate

Analytical Method: Quantitative

Specificity: Cross Reactivity: This ELISA kit shows no cross-reactivity with any of the cytokines tested: Human Angiogenin, BDNF, BLC, BMP-6, BMP-7, ENA-78, FGF-4, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, I-309, IP-10, G-CSF, GM-CSF, IFN-gamma, Leptin, MCP-1, MCP-2, MCP-3, MDC, MIP-1 alpha, MIP-1 beta, MIP-1 delta, PARC, PDGF, RANTES, SCF, TARC, TGF-beta, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.

Sensitivity: 15 pg/mL

Characteristics:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Components:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Material not included:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis
• Cell lysate buffer

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Reagent Preparation:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: Tissue lysate and cell lysate sample should be diluted at least 5-folds with 1x Sample Diluent Buffer.
   3. Sample Diluent Buffer (Item D) and Assay Diluent (Item E) should be diluted 5-folds with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C. Add 400 µL 1x Sample Diluent Buffer (Item D) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 80 µL BMP-4 standard from the vial of Item C, into a tube with 586.7 µL Sample Diluent Buffer to prepare a 6,000 pg/mL stock standard solution. Pipette 400 µL 1x Sample Diluent Buffer into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. 1x Sample Diluent Buffer serves as the zero standard (0 pg/mL). 200 µL 200 µL 200 µL 200myl 200 µL 200 µL 80 µL standard + 586.7 µL 6,000 2,000 666.7 222.2 74.07 24.69 8.23 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diuent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 65-foldss with 1x Assay Diuent and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) before use. HRP-Streptavidin concentrate should be diluted 80-fold with 1x Assay Diuent. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 125 µL of HRP-Streptavidin concentrate into a tube with 10 ml 1x Assay Diluent to prepare a final 80 fold diluted HRP-Streptavidin solution. Mix well.
   8. Cell lysate buffer (Item J) should be diluted 2-fold with deionized or distilled water (for cell lysate and tissue lysate).

Assay Procedure:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well with plate holder and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. 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 paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Calculation of Results:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent Human BMP-4 concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 n m 0.001 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of BMP-4 is typically less than 15 pg/mL.
Recovery: Recovery was determined by spiking various levels of human BMP-4 into human tissue lysate and cell lysate. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Tissue lysate 89.47 78-102 Cell lysate 92.45 80-103
Linearity: Sample Type Tissue Cell Lysate lysate 1:2 Average % of 84.31 85.75 Expected Range ( %) 77-103 77-102 1:4 Average % of 86.78 88.54 Expected Range ( %) 79-102 80-104
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Assay Precision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Handling

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Expiry Date: 6 months

References for BMP4 ELISA Kit (ABIN624951)

Wozney: "The bone morphogenetic protein family and osteogenesis." in: Molecular reproduction and development, Vol. 32, Issue 2, pp. 160-7, (1992) (PubMed).

Aldinger, Herr, Küsswetter, Reis, Thielemann, Holz: "Bone morphogenetic protein: a review." in: International orthopaedics, Vol. 15, Issue 2, pp. 169-77, (1991) (PubMed).

Target Details for BMP4

Target: BMP4 (Bone Morphogenetic Protein 4 (BMP4))

Alternative Name: BMP4 (BMP4 Products)

Synonyms: BMP2B ELISA Kit, BMP2B1 ELISA Kit, MCOPS6 ELISA Kit, OFC11 ELISA Kit, ZYME ELISA Kit, Bmp-4 ELISA Kit, Bmp2b ELISA Kit, Bmp2b-1 ELISA Kit, Bmp2b1 ELISA Kit, BOMPR4A ELISA Kit, bmp-4 ELISA Kit, zbmp-4 ELISA Kit, zgc:100779 ELISA Kit, BMP-4 ELISA Kit, XBMP-4 ELISA Kit, bmp2b ELISA Kit, bmp2b1 ELISA Kit, bmp4 ELISA Kit, ofc11 ELISA Kit, xbmp4 ELISA Kit, zyme ELISA Kit, BMP4 ELISA Kit, bone morphogenetic protein 4 ELISA Kit, bone morphogenetic protein 4 L homeolog ELISA Kit, bone morphogenetic protein 4 S homeolog ELISA Kit, BMP4 ELISA Kit, Bmp4 ELISA Kit, bmp4 ELISA Kit, bmp4.L ELISA Kit, bmp4.S ELISA Kit

Background: The Human BMP-4 ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human BMP-4 cell lysate and tissue lysate. This assay employs an antibody specific for human BMP-4 coated on a 96-well plate. Standards and samples are pipetted into the wells and BMP-4 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human BMP-4 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of BMP-4 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.

Gene ID: 652

UniProt: P12644

Pathways: Steroid Hormone Mediated Signaling Pathway, Regulation of Muscle Cell Differentiation, Tube Formation, Skeletal Muscle Fiber Development