BMP4 ELISA Kit

Catalog No. ABIN624950

This Colorimetric ELISA kit is designed for the quantitative measurement of Human BMP4. There are 11 publications available.

Quick Overview for BMP4 ELISA Kit (ABIN624950)

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

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 15 pg/mL - 6000 pg/mL

Application: ELISA

Sample Type: Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Lysate

Product Details BMP4 ELISA Kit

Minimum Detection Limit: 15 pg/mL

Purpose: Human BMP-4 ELISA Kit for cell culture supernatants, plasma, serum, and lysate samples.

Analytical Method: Quantitative

Specificity: 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, GMCSF, 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.

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

,

• 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

Application Details

Application Notes: Suggested Plasma Dilution: 2 fold

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: If your samples need to be diluted, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. 1x Assay Diluent B (Item E) should be used for dilution of culture supernantants and urine. Suggested dilution for normal serum/plasma: 2 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent B should be diluted 5-fold with deionized or distilled water.
   4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium and urine) 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 Assay Diluent A or 1x Assay Diluent B to prepare a 6,000 pg/mL stock standard solution. Pipette 400 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 200 µL 200myl 200 µL 200 µL 200 µL 200 µL 80 µL standard +586.7 µL 6000 2000 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 Diluent B 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-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 80-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 150 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a final 80 fold diluted HRP-Streptavidin solution. Mix well.

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 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 A Human BMP-4 concentration (pg/mL) 10 100 1000 10000 O D =4 50 n m 0.01 0.1 1 10 Assay Diluent B Human BMP-4 concentration (pg/mL) 10 100 1000 10000 O D =4 50 n m 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 serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 92.95 80-101 Plasma 94.32 82-102 Cell culture media 98.25 85-104
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 94 93 95 Range ( %) 83-103 82-102 84-103 1:4 Average % of Expected 96 95 101 Range ( %) 85-104 83-102 88-106 1:8 Average % of Expected 96 97 94 Range ( %) 84-104 85-104 84-103
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 (ABIN624950)

Bhattacharyya, Feferman, Tobacman: "Regulation of chondroitin-4-sulfotransferase (CHST11) expression by opposing effects of arylsulfatase B on BMP4 and Wnt9A." in: Biochimica et biophysica acta, Vol. 1849, Issue 3, pp. 342-52, (2015) (PubMed).

Pallotta, Sun, Wrona, Freytes: "BMP protein-mediated crosstalk between inflammatory cells and human pluripotent stem cell-derived cardiomyocytes." in: Journal of tissue engineering and regenerative medicine, (2015) (PubMed).

Youn, Zhou, Cai: "Bone Morphogenic Protein 4 Mediates NOX1-Dependent eNOS Uncoupling, Endothelial Dysfunction, and COX2 Induction in Type 2 Diabetes Mellitus." in: Molecular endocrinology (Baltimore, Md.), Vol. 29, Issue 8, pp. 1123-33, (2015) (PubMed).

Zhang, Chen, Guo, Yuan, Zhang, Xu, Nemeth, Ganz, Liu: "Disordered hepcidin-ferroportin signaling promotes breast cancer growth." in: Cellular signalling, Vol. 26, Issue 11, pp. 2539-50, (2014) (PubMed).

Dincel, Sepici-Dincel: "The importance and the differences of bone morphogenetic proteins for osteoporotic hip fractures." in: Acta orthopaedica Belgica, Vol. 80, Issue 2, pp. 216-21, (2014) (PubMed).

Lai, Yang: "BMP4 is a novel transcriptional target and mediator of mammary cell migration downstream of the Hippo pathway component TAZ." in: Cellular signalling, Vol. 25, Issue 8, pp. 1720-8, (2013) (PubMed).

Lee, Cheng, Bilen, Lu, Satcher, Yu-Lee, Gallick, Maity, Lin: "BMP4 promotes prostate tumor growth in bone through osteogenesis." in: Cancer research, Vol. 71, Issue 15, pp. 5194-203, (2011) (PubMed).

Mi, Lee, Fu, Chow, Lin: "Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer." in: Gene therapy, Vol. 18, Issue 5, pp. 452-61, (2011) (PubMed).

Wahlström, Linder, Ansell, Kalén, Söderström, Magnusson: "Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis." in: Platelets, Vol. 22, Issue 6, pp. 452-60, (2011) (PubMed).

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

Background:

Gene Symbols: BMP4, BMP2B, DVR4

Protein Name (Synonyms): Bone morphogenetic protein 4 (BMP-4) (Bone morphogenetic protein 2B) (BMP-2B)

Gene ID: 652

UniProt: P12644

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