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Human Adipokine Array Q1

Details for Product No. ABIN625694, Supplier: Log in to see
Methode Type
Sandwich ELISA
Antibody Array (AA), Multiplex ELISA (mpELISA)
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Purpose Quantibody® Human Adipokine Array 1, multiplex ELISA array Kit for quantitative measurement of 10 Human Adipokines in 8-10 samples. Suitable for all sample Types
Brand Quantibody®
Sample Type Serum, Plasma, Cell Culture Supernatant, Cell Lysate, Tissue Lysate, Biological Fluids
ProductDetails: Analytical Method Quantitative
Specificity Quantibody® Human Adipokine Array 1 detects: IGF-I, IL1alpha, interleukin-6, interleukin-8, Insulin, Leptin, MCP-1, PAI-1, Resistin, TNF alpha
Components - Quantibody® Array Slide
- Blocking Buffer
- Wash Buffer 1
- Wash Buffer 2
- Lyophilized Standard Mix
- Biotinylated Detection Antibody Cocktail
- Streptavidin-Conjugated Fluor
- 30 ml Centrifuge Tube
- Adhesive Plastic Strips
- Manual
Material not included Distilled or deionized water
Small plastic boxes or containers
Pipettors, pipette tips and other common lab consumables
Orbital shaker or oscillating rocker
Aluminum foil
Gene microarray scanner or similar laser fluorescence scanner
Application Notes Completely cover array area with sample or buffer during incubation. Avoid foaming during incubation steps. Perform all incubation and wash steps under gentle rocking or rotation. Cover the incubation chamber with adhesive film during incubation, particularly when incubation is more than 2 hours or <70 µl of sample or reagent is used. Several incubation steps such as step 6 (blocking), step 7 (sample incubation), step 10 (detection antibody incubation), or step 13 (Cy3 equivalent dyestreptavidin incubation) may be done overnight at 4°C. Please make sure to cover the incubation chamber tightly to prevent evaporation.

The Quantibody arrays are quantitative multiplex ELISA arrays featuring fluorescent detection. The antibodies are spotted on glass slide solid supports and require a laser scanner for data collection. Cytokine standards are provided with the array for calculation of target protein concentrations. All Quantibody arrays feature the sandwich immunoassay principle, utilizing an immobilized capture antibody along with a corresponding biotinylated detection antibody.

Sample Volume 50 μL
Assay Time 6 h
Plate Glass Slide
Protocol - Dry the glass slide
- Prepare Standards
- Block array surface
- Incubate with Samples and Standards
- Incubate with Biotinylated Detection Antibody Cocktail
- Incubate with Streptavidin-Conjugated Fluor
- Disassemble the glass slide
- Scan with a gene microarray laser scanner
- Perform densitometry and analysis
Sample Preparation

Use serum-free conditioned media if possible. If serum-containing conditioned media is required, it is highly recommended that complete medium be used as a control since many types of sera contains cytokines. We recommend the following parameters for your samples: 50 to 100 l of original or diluted serum, plasma, cell culture media, or other body fluid, or 50-500 g/ml of protein for cell and tissue lysates. If you experience high background or the readings exceed the detection range, further dilution of your sample is recommended.

Assay Procedure
  1. Take out the glass slide from the box, and let it equilibrate to room temperature inside the sealed plastic bag for 20-30 minutes. Remove slide from the plastic bag, peel off the cover film, and let it air dry for another 1-2 hours.
    2. Reconstitute the Cytokine Standard Mix (lyophilized) by adding 500 µl Sample Diluent to the tube. For best recovery, always quick-spin vial prior to opening. Dissolve the powder thoroughly by a gentle mix. Labeled the tube as Std1.
    3. Label 6 clean microcentrifuge tubes as Std2 to Std7. Add 200 µl Sample Diluent to each of the tubes.
    4. Pipette 100 µl Std1 into tube Std2 and mix gently. Perform 5 more serial dilutions by adding 100 µl Std2 to tube Std3 and so on.
    5. Add 100 µl Sample Diluent to another tube labeled as CNTRL. Do not add standard cytokines or samples to the CNTRL tube, which will be used as negative control. For best results, include a set of standards in each slide.
    6. Add 100 µl Sample Diluent into each well and incubate at room temperature for 30 minutes to block slides.
    7. Decant buffer from each well. Add 100 µl standard cytokines or samples to each well. Incubate arrays at room temperature for 1-2 hour.
    8. Wash:
    - Decant the samples from each well, and wash 5 times (5 min each) with 150 µl of 1X Wash Buffer I at room temperature with gentle rocking. Completely remove wash buffer in each wash step. Dilute 20x Wash Buffer I with H2O.
    - Decant the 1x Wash Buffer I from each well, wash 2 times (5 min each) with 150 µl of 1X Wash Buffer II at room temperature with gentle rocking. Completely remove wash buffer in each wash step. Dilute 20X Wash Buffer II with H2O.
    9. Reconstitute the detection antibody by adding 1.4 ml of Sample Diluent to the tube. Spin briefly.
    10. Add 80 µl of the detection antibody cocktail to each well. Incubate at room temperature for 1-2 hour.
    11. Decant the samples from each well, and wash 5 times (5 mins each) with 150 µl of 1X Wash Buffer I and then 2 times with 150 µl of 1x Wash Buffer II at room temperature with gentle rocking. Completely remove wash buffer in each wash step.
    12. After briefly spinning down, add 1.4 ml of Sample Diluent to Cy3 equivalent dye-conjugated streptavidin tube. Mix gently.
    13. Add 80 µl of Cy3 equivalent dye-conjugated streptavidin to each well. Cover the device with aluminum foil to avoid exposure to light or incubate in dark room. Incubate at room temperature for 1 hour.
    14. Decant the samples from each well, and wash 5 times (5 mins each) with 150 µl of 1X Wash Buffer I at room temperature with gentle rocking. Completely remove wash buffer in each wash step.
    15. Disassemble the device by pushing clips outward from the slide side. Carefully remove the slide from the gasket.
    16. Place the slide in the Slide Washer/Dryer (a 4-slide holder/centrifuge tube), add enough 1x Wash Buffer I (about 30 ml) to cover the whole slide, and then gently shake at room temperature for 15 minutes. Decant Wash Buffer I. Wash with 1x Wash Buffer II (about 30 ml) and gently shake at room temperature for 5 minutes.
    17. Remove water droplets completely by gently applying suction with a pipette to remove water droplets. Do not touch the array, only the sides.
    18. Imaging: The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength (green channel) such as Axon GenePix. Make sure that the signal from the well containing the highest standard concentration (Std1) receives the highest possible reading, yet remains unsaturated.
Calculation of Results

Data extraction can be done using the GAL file that is specific for this array along with the microarray analysis software (GenePix, ScanArray Express, ArrayVision, MicroVigene, etc.).

Assay Precision Reproducibility: CV < 20%
Restrictions For Research Use only
Handling Advice Do not touch the surface of the slides, as the microarray slides are very sensitive. Hold the slides by the edges only. Handle all buffers and slides with powder free gloves. Handle glass slide/s in clean environment. The Quantibody slides do not have bar codes. To help distinguish one slide from another, transcribe the slide serial number from the slide bag to the back of the slide with an ultra-fine point permanent marker. Please Note:Red permanent marker can significantly interfere with fluorescent signal detection. We recommend marking your slides with a green, blue or black ultra-fine point permanent marker. Please write the number on the very bottom edge of the slide. Do not write on the arrayed well areas.
Storage -20 °C
Storage Comment Upon receipt, all components should be stored at -20°C. The kit will retain activity for up to 6 months. Once thawed, the glass slide, standard mix, antibody cocktail and dye-conjugated Streptavidin should be kept at -20°C. All other components may be stored at 4°C. The entire kit should be used within 6 months of purchase.
Expiry Date 6 months
Product cited in: Lin, Chen, Chen et al.: "Adipocytes modulate the electrophysiology of atrial myocytes: implications in obesity-induced atrial fibrillation." in: Basic research in cardiology, Vol. 107, Issue 5, pp. 293, 2012 (PubMed).