AXL ELISA Kit

Catalog No. ABIN624944

Product Details AXL ELISA Kit

Target: AXL (AXL Receptor tyrosine Kinase (AXL))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 2-2000 pg/mL

Minimum Detection Limit: 2 pg/mL

Application: ELISA

Purpose: Human AXL ELISA Kit for cell culture supernatants, plasma, and serum samples.

Sample Type: Plasma, Cell Culture Supernatant, Serum

Analytical Method: Quantitative

Specificity: This ELISA kit shows no cross-reactivity with any of the cytokines tested: human Angiogenin, BDNF, BLC, 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: < 2 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

Application Details

Application Notes: Recommended Dilution for serum and plasma samples20 - 200 fold

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: 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: 20-200 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 100 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 20 µL Axl standard from the vial of Item C, into a tube with 980 µL Assay Diluent A or 1x Assay Diluent B to prepare a 2,000 pg/mL 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 20 µL standard + 980 µL 2000 666.7 222.2 74.07 24.69 8.23 2.74 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 80-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 300-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 50 µL of HRP-Streptavidin concentrate into a tube with 15 ml 1x Assay Diluent B to prepare a final 300 fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). 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 Axl concentration (pg/mL) O D =4 50 n m 0.001 0.01 0.1 1 10 0 10 100 1,000 10,000 Assay Diluent B Human Axl concentration (pg/mL) O D =4 50 n m 0.001 0.01 0.1 1 10 0 10 100 1,000 10,000
Sensitivity: The minimum detectable dose of Axl is typically less than 2 pg/mL.
Recovery: Recovery was determined by spiking various levels of human Axl into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 91.77 82-102 Plasma 93.49 83-103 Cell culture media 92.35 82-102
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 93 94 94 Range ( %) 83-103 82-102 84-103 1:4 Average % of Expected 94 92 96 Range ( %) 84-104 83-103 83-103
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 AXL ELISA Kit (ABIN624944)

Orme, Du, Vanarsa, Mayeux, Li, Mutwally, Arriens, Min, Hutcheson, Davis, Chong, Satterthwaite, Wu, Mohan: "Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE." in: Clinical immunology (Orlando, Fla.), Vol. 169, pp. 58-68, (2017) (PubMed).

Wu, Ding, Han, Arriens, Wei, Han, Pedroza, Jiang, Anolik, Petri, Sanz, Saxena, Mohan: "Antibody-Array-Based Proteomic Screening of Serum Markers in Systemic Lupus Erythematosus: A Discovery Study." in: Journal of proteome research, Vol. 15, Issue 7, pp. 2102-14, (2016) (PubMed).

Liu, Yang, Zuo, Xiao, Chen, Liu: "Elevated serum levels of soluble Axl in acute coronary syndrome." in: The American journal of the medical sciences, Vol. 349, Issue 2, pp. 124-9, (2015) (PubMed).

Hsiao, Lin, Hsieh, Chu, Shieh, Hsieh, Lee, Hung: "Circulating growth arrest-specific 6 protein is associated with adiposity, systemic inflammation, and insulin resistance among overweight and obese adolescents." in: The Journal of clinical endocrinology and metabolism, Vol. 98, Issue 2, pp. E267-74, (2013) (PubMed).

Target Details for AXL

Target: AXL (AXL Receptor tyrosine Kinase (AXL))

Alternative Name: AXL (AXL Products)

Synonyms: JTK11 ELISA Kit, UFO ELISA Kit, AI323647 ELISA Kit, Ark ELISA Kit, Tyro7 ELISA Kit, Ufo ELISA Kit, AXL receptor tyrosine kinase ELISA Kit, Axl receptor tyrosine kinase ELISA Kit, AXL ELISA Kit, Axl ELISA Kit

Background: The Human Axl ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human Axl in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human Axl coated on a 96-well plate. Standards and samples are pipetted into the wells and Axl present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human Axl 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 Axl 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: 558

UniProt: P30530

Pathways: RTK Signaling, Cellular Response to Molecule of Bacterial Origin