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|Application / Reactivity||Human|
|ELISA||17 ELISA Kits|
|Antigen||Fibroblast Growth Factor 4 (FGF4) ELISA Kits|
Kits with alternative reactivity to:
|Methode Type||Sandwich ELISA|
|Detection Range||50-18000 pg/mL|
|Minimum Detection Limit||50 pg/mL|
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Product Details FGF4 ELISA KitTarget details Application Details Handling Images
|Purpose||Human FGF-4 ELISA Kit for cell culture supernatants, plasma, and serum samples.|
|Sample Type||Plasma, Cell Culture Supernatant, Serum|
|Specificity||This ELISA kit shows no cross-reactivity with any of the cytokines tested: Human Angiogenin, BDNF, BLC, ENA-78, FGF-6, FGF-7, FGF-9, 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||< 50 pg/mL|
|Material not included||
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|Alternative Name||FGF-4 (FGF4 ELISA Kit Abstract)|
|Background||FGF (Fibroblast growth factors) constitutes a family of related 16-18 kDa proteins controlling normal growth and differentiation of mesenchymal, epithelial, and neuroectodermal cell types. FGF-4 has potent transforming potential, apparently through an autocrine mechanism of action, and it is a potent angiogenic factor. It has also been extensively studied for its effects in embryogenesis The Human FGF-4 ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human FGF-4 in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human FGF-4 coated on a 96-well plate. Standards and samples are pipetted into the wells and FGF-4 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human FGF-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 FGF-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%.|
|Pathways||RTK Signaling, Fc-epsilon Receptor Signaling Pathway, EGFR Signaling Pathway, Neurotrophin Signaling Pathway, Stem Cell Maintenance|
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|Application Notes||Recommended Dilution for serum and plasma samples2 fold|
|Sample Volume||100 μL|
|Plate||Pre-coated,Strips (12 x 8)|
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 180 µL FGF-4 standard from the vial of Item C, into a tube with 320 µL Assay Diluent A or 1x Assay Diluent B to prepare a 18,000 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 180 µL standard + 320 µL 200myl 200 µL 200 µL 200 µL 200 µL 18,000 6,000 2,000 666.7 222.2 74.07 24.69 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 120-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 100 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a final 120 fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). Mix well.
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 FGF-4 concentration (pg/mL) O D =4 50 (n m ) 0.001 0.01 0.1 1 10 10 100 1,000 10,000 100,000 Assay Diluent B Human FGF-4 concentration (pg/mL) O D =4 50 (n m ) 0.01 0.1 1 10 10 100 1,000 10,000 100,000
Sensitivity: The minimum detectable dose of FGF-4 is typically less than 50 pg/mL.
Recovery: Recovery was determined by spiking various levels of human FGF-4 into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 97.57 89-112 Plasma 96.21 88-110 Cell culture media 91.54 80-103
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 93 94 95 Range ( %) 84-103 84-105 85-106 1:4 Average % of Expected 92 92 96 Range ( %) 83-104 85-104 84-105 1:8 Average % of Expected 95 94 95 Range ( %) 82-105 83-105 87-103
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %
|Assay Precision||Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %|
|Restrictions||For Research Use only|
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|Handling Advice||Avoid repeated freeze-thaw cycles.|
|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|
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