CY-8052 Dog Adiponectin ELISA Kit

CY-8052 Dog Adiponectin ELISA Kit
Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
ELISA Kit for Measuring Dog Adiponectin
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CircuLex Dog Adiponectin ELISA Kit
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Intended Use................................................ 1
Storage......................................................... 1
Introduction.................................................. 2
Principle of the Assay.................................. 3
Materials Provided....................................... 4
Materials Required but not Provided........... 5
Precautions and Recommendations............. 6
Sample Collection and Storage.....................7
Detailed Protocol......................................... 8-9
Calculations.................................…............ 10
Measurement Range................................... 10
Troubleshooting.......................................... 10
Reagent Stability......................................... 11
Assay Characteristics.................................. 11-13
Example of Test Results............................. 13-14
References................................................... 15
Related Products......................................... 15
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Intended Use
The CycLex Research Product CircuLex Dog Adiponectin ELISA Kit is used for the quantitative
measurement of dog adiponectin in serum, plasma, tissue culture medium and other biological media.
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This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.
Storage
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• Upon receipt store all components at 4°C.
• Don’t expose reagents to excessive light.
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Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
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Introduction
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Adiponectin, also referred as Acrp30, AdipoQ and GBP-28, is a recently discovered 244 amino acid
protein, the product of the apM1 gene, which is physiologically active and specifically and highly
expressed in adipose cells (adipokine). The protein belongs to the soluble defense collagen superfamily;
it has a collagen-like domain structurally homologous with collagen VIII and X and complement factor
C1q-like globular domain (1, 2). Adiponectin forms homotrimers, which are the building blocks for
higher order complexes found circulating in serum (3, 4). Circulating adiponectin levels are high (5-30
g/mL), accounting for approximately 0.01% of total plasma protein (5-8). Adiponectin receptors
AdipoR1 and AdipoR2 have been recently cloned; AdipoR1 is abundantly expressed in skeletal muscle,
whereas AdipoR2 is predominantly expressed in the liver (9). Paradoxically, adipose tissue-expressed
adiponectin levels are inversely related to the degree of adiposity (10,11). A reduction in adiponectin
serum levels is accompanied by insulin resistance states, such as obesity and type 2 diabetes mellitus (12,
13). It is also reported in patients with coronary artery disease (13). Increased adiponectin levels are
associated with type 1 diabetes mellitus, anorexia nervosa and chronic renal failure. Adiponectin
concentrations correlate negatively with glucose, insulin, triglyceride concentrations and body mass
index and positively with high-density lipoprotein-cholesterol levels and insulin-stimulated glucose
disposal. Adiponectin has been shown to increase insulin sensitivity and decrease plasma glucose by
increasing tissue fat oxidation. It inhibits the inflammatory processes of atherosclerosis suppressing the
expression of adhesion and cytokine molecules in vascular endothelial cells and macrophages,
respectively. This adipokine plays a role as a scaffold of newly formed collagen in myocardial
remodeling after ischemic injury and also stimulates angiogenesis by promoting cross-talk between
AMP-activated protein kinase and Akt signaling in endothelial cells (14).
Injection of adiponectin into non-obese diabetic mice leads to an insulin-independent decrease in
glucose levels (15). This is likely due to insulin-sensitizing effects involving adiponectin-regulation of
triglyceride metabolism (15). A truncated form of adiponectin (gAdiponectin) containing only the
C-terminal globular domain has been identified in the blood, and recombinant gAdiponectin has been
shown to regulate weight reduction as well as free fatty acid oxidation in mouse muscle and liver (16,
17). The full-length recombinant adiponectin protein is apparently less potent at mediating these effects
(16, 17).
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Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
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Principle of the Assay
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The CircuLex Dog Adiponectin ELISA Kit employs the quantitative sandwich enzyme immunoassay
technique. An antibody specific for adiponectin has been pre-coated onto a microplate. Standards and
samples are pipetted into the wells and the immobilized antibody binds any adiponectin present. After
washing away any unbound substances, an HRP conjugated antibody specific for adiponectin is added to
the wells. Following a wash to remove any unbound antibody HRP conjugate, the remaining conjugate is
allowed to react with the substrate H2O2-tetramethylbenzidine. The reaction is stopped by addition of
acidic solution and absorbance of the resulting yellow product is measured at 450 nm. The absorbance is
proportional to the concentration of adiponectin. A standard curve is constructed by plotting absorbance
values versus adiponectin concentrations of calibrators, and concentrations of unknown samples are
determined using this standard curve.
The CircuLex Dog Adiponectin ELISA Kit is designed to measure the concentration of dog
adiponectin from dog serum/plasma, adipocytes, or conditioned medium.
Summary of Procedure
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Add 100 µL of diluted sample to the wells
Incubate for 1 hour at room temp.
Wash the wells
Add 100 µL of HRP conjugated anti-adiponectin antibody
Incubate for 1hour at room temp.
Wash the wells
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Add 100 µL of Substrate Reagent
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Add 100 µL of Stop Solution
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Measure absorbance at 450 nm
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User’s Manual
For Research Use Only, Not for use in diagnostic procedures
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Materials Provided
All samples and standards should be assayed in duplicate. The following components are supplied and
are sufficient for the one 96-well microplate kit.
Microplate: One microplate supplied ready to use, with 96 wells (12 strips of 8-wells) in a foil, zip-lock
bag with a desiccant pack. Wells are coated with anti-adiponectin antibody as a capture antibody.
10X Wash Buffer: One bottle containing 100 mL of 10X buffer containing 2 %Tween®-20
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Dilution Buffer: One bottle containing 50 mL of 1X buffer; use for sample dilution. Ready to use.
Dog Adiponectin Standard: One vial containing 48 ng of lyophilized dog adiponectin
HRP conjugated Detection Antibody: One bottle containing 12 mL of HRP (horseradish peroxidase)
conjugated anti-adiponectin antibody. Ready to use.
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Substrate Reagent: One bottle containing 20 mL of the chromogenic substrate, tetra-methylbenzidine
(TMB). Ready to use.
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Stop Solution: One bottle containing 20 mL of 1 N H2SO4. Ready to use.
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Materials Required but not Provided
• Pipettors: 2-20 µL, 20-200 µL and 200-1000 µL precision pipettors with disposable tips.
• Precision repeating pipettor
• Orbital microplate shaker
• Microcentrifuge and tubes for sample preparation.
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• Vortex mixer
• Microplate washer: optional (Manual washing is possible but not preferable)
• Plate reader capable of measuring absorbance in 96-well plates at dual wavelengths of 450 nm/540
nm. Dual wavelengths of 450/550 or 450/595 nm can also be used. The plate can also be read at a
single wavelength of 450 nm, which will give a somewhat higher reading.
• 500 or 1000 mL graduated cylinder
• Reagent reservoirs
• Deionized water of the highest quality
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• Disposable paper towels
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• Software package facilitating data generation and analysis :optional
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Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
Precautions and Recommendations
• Allow all the components to come to room temperature before use.
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• All microplate strips that are not immediately required should be returned to the zip-lock pouch, which
must be carefully resealed to avoid moisture absorption.
• Do not use kit components beyond the indicated kit expiration date.
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• Use only the microtiter wells provided with the kit.
• Rinse all detergent residue from glassware.
• Use deionized water of the highest quality.
• Do not mix reagents from different kits.
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• The buffers and reagents in this kit may contain preservatives or other chemicals. Care should be taken
to avoid direct contact with these reagents.
• Do not mouth pipette or ingest any of the reagents.
• Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are
handled.
• Dispose of tetra-methylbenzidine (TMB) containing solutions in compliance with local regulations.
• Avoid contact with Substrate Solution which contains hydrogen peroxide.
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• Avoid contact with Stop Solution which contains Sulfuric Acid.
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• Wear gloves and eye protection when handling immunodiagnostic materials and samples of dog origin,
and these reagents. In case of contact with the Stop Solution and the Substrate Solution, wash skin
thoroughly with water and seek medical attention, when necessary.
• Biological samples might be contaminated with infectious agents. Do not ingest, expose to open
wounds or breathe aerosols. Wear protective gloves and dispose of biological samples properly.
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• CAUTION: Sulfuric Acid is a strong acid. Wear disposable gloves and eye protection when
handling Stop Solution.
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User’s Manual
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Sample Collection and Storage
Serum: Allow blood samples to clot for 60 ± 30 minutes. Centrifuge the samples at 4°C for 10 minutes
at 1,000 x g. Remove serum and assay immediately or store samples on ice for up to 6 hours before
assaying. Aliquots of serum may also be stored at below -70°C for extended periods of time. Avoid
repeated freeze-thaw cycles.
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Plasma: Collect plasma using EDTA-Na2 as the anticoagulant. If possible, collect the plasma into a
mixture of EDTA-Na2 and Futhan5 to stabilize the sample against spontaneous in vitro complement
activation. Immediately centrifuge samples at 4°C for 15 minutes at 1,000 x g. Assay immediately or
store samples on ice for up to 6 hours before assaying. Aliquots of plasma may also be stored at below
-70°C for extended periods of time. Avoid repeated freeze-thaw cycles.
Note: Citrate plasma has not been validated for use in this assay.
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Other biological samples: Remove any particulates by centrifugation and assay immediately or aliquot
and store samples at below -70°C. Avoid repeated freeze-thaw cycles.
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Dog Adiponectin ELISA Kit
User’s Manual
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Detailed Protocol
The CycLex Research Product CircuLex Dog Adiponectin ELISA Kit is provided with removable
strips of wells so the assay can be carried out on separate occasions using only the number of strips
required for the particular determination. Since experimental conditions may vary, an aliquot of the Dog
Adiponectin Standard within the kit, should be included in each assay as a calibrator. Disposable pipette
tips and reagent troughs should be used for all liquid transfers to avoid cross-contamination of reagents
or samples.
Preparation of Working Solutions
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All reagents need to be brought to room temperature prior to the assay. Assay reagents are supplied
ready-to-use, with the exception of 10X Wash Buffer and Dog Adiponectin Standard.
1. Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of
deionized (distilled) water (ddH2O). Mix well. Store at 4°C for two weeks or -20°C for long-term
storage.
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2. Reconstitute Dog Adiponectin Standard with 1.0 mL of ddH2O. The concentration of the dog
Adiponectin in vial should be 48 ng/mL, which is referred as a Master Standard of dog
Adiponectin.
Prepare Standard Solutions as follows:
Use the Master Standard to produce a dilution series (below). Mix each tube thoroughly before
the next transfer. The 12 ng/mL standard (Std.1) serves as the highest standard. The Dilution
Buffer serves as the zero standard (Blank).
Volume of Standard
150 µL of Master Standard
300 µL of Std. 1 (12.00 ng/ml)
300 µL of Std. 2 (6.00 ng/ml)
300 µL of Std. 3 (3.00 ng/ml)
300 µL of Std. 4 (1.50 ng/ml)
300 µL of Std. 5 (0.75 ng/ml)
300 µL of Std. 6 (0.38 ng/ml)
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Std.1
Std.2
Std.3
Std.4
Std.5
Std.6
Std.7
Blank
-
Dilution Buffer
450 µL
300 µL
300 µL
300 µL
300 µL
300 µL
300 µL
300 µL
Concentration
12.00 ng/mL
6.00 ng/mL
3.00 ng/mL
1.50 ng/mL
0.75 ng/mL
0.38 ng/mL
0.19 ng/mL
0 ng/mL
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Note: Do not use a Repeating pipette. Change tips for every dilution. Wet tip with Dilution Buffer
before dispensing. Unused portions of Master Standards should be aliquoted and stored at below
-70°C immediately. Avoid multiple freeze and thaw cycles.
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Sample Preparation
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• Serum and plasma samples require a 5,000-fold dilution.
e.g. Step One : Dilute samples 1:100 with Dilution Buffer
(2 µL of sample + 198 µL of Dilution Buffer)
Step Two: Dilute Step one samples 1:50 with Dilution Buffer
(5 µL of Step One samples + 245 µL of Dilution Buffer)
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Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
Assay Procedure
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1. Remove the appropriate number of microtiter wells from the foil pouch and place them into the well
holder. Return any unused wells to the foil pouch, refold, seal with tape and store at 4°C.
2. Dilute samples with Dilution Buffer. (See “Sample Preparation” above.)
3. Pipette 100 µL of Standard Solutions (Std1-Std7, Blank) and diluted samples in duplicates, into
the appropriate wells.
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4. Incubate the plate at room temperature (ca.25°C) for 1 hour, shaking at ca. 300 rpm on an orbital
microplate shaker.
5. Wash 4-times by filling each well with Wash Buffer (350 µL) using a squirt bottle, multi-channel
pipette, manifold dispenser or microplate washer.
6. Add 100 µL of HRP conjugated Detection Antibody into each well.
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7. Incubate the plate at room temperature (ca.25°C) for 1 hour, shaking at ca. 300 rpm on an orbital
microplate shaker.
8. Wash 4-times by filling each well with Wash Buffer (350 µL) using a squirt bottle, multi-channel
pipette, manifold dispenser or microplate washer.
9. Add 100 µL of Substrate Reagent. Avoid exposing the microtiter plate to direct sunlight. Covering
the plate with e.g. aluminum foil is recommended. Return Substrate Reagent to 4°C immediately after
the necessary volume is removed.
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10. Incubate the plate at room temperature (ca. 25°C) for 10-20 minutes, shaking at ca. 300 rpm on an
orbital microplate shaker. The incubation time may be extended up to 30 minutes if the reaction
temperature is below than 20°C.
11. Add 100 µL of Stop Solution to each well in the same order as the previously added Substrate
Reagent.
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12. Measure absorbance in each well using a spectrophotometric microplate reader at dual wavelengths of
450/540 nm. Dual wavelengths of 450/550 or 450/595 nm can also be used. Read the microplate at
450 nm if only a single wavelength can be used. Wells must be read within 30 minutes of adding the
Stop Solution.
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Note-1: 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.
Note-2: Reliable standard curves are obtained when either O.D. values do not exceed 0.2 units for the
blank (zero concentration), or 2.5 units for the highest standard concentration. The plate
should be monitored at 5-minute intervals for approximately 30 minutes.
Note-3: If the microplate reader is not capable of reading absorbance greater than the absorbance of the
highest standard, perform a second reading at 405 nm. A new standard curve, constructed
using the values measured at 405 nm, is used to determine Adiponectin concentration of
off-scale samples. The readings at 405 nm should not replace the on-scale readings at 450 nm.
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Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
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Calculations
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Average the duplicate readings for each standard, control, and sample and subtract the average zero
standard optical density. Plot the optical density for the standards versus the concentration of the
standards and draw the best curve. The data can be linearized by using log/log paper and regression
analysis may be applied to the log transformation. To determine the Adiponectin concentration of each
sample, first find the absorbance value on the y-axis and extend a horizontal line to the standard curve.
At the point of intersection, extend a vertical line to the x-axis and read the corresponding Adiponectin
concentration. If the samples have been diluted, the concentration read from the standard curve must be
multiplied by the dilution factor.
1. The dose-response curve of this assay fits best to a sigmoidal 5-parameter logistic equation. The
results of unknown samples can be calculated with any computer program having a 5-parameter
logistic function. It is important to make an appropriate mathematical adjustment to accommodate for
the dilution factor.
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2. Most microtiter plate readers perform automatic calculations of analyte concentration. The calibration
curve is constructed by plotting the absorbance (Y) of calibrators versus log of the known
concentration (X) of calibrators, using the four-parameter function. Alternatively, the logit log
function can be used to linearize the calibration curve (i.e. logit of absorbance (Y) is plotted versus log
of the known concentration (X) of calibrators).
3. Dilution factors need to be taken into consideration in calculating the Adiponectin concentration.
Measurement Range
Troubleshooting
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The measurement range is 0.19 ng/mL to 12 ng/mL. Any sample reading higher than the highest
standard should be diluted with Dilution Buffer in higher dilution and re-assayed. Dilution factors need
to be taken into consideration in calculating the dog adiponectin concentration.
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1. The Dog Adiponectin Standard should be run in duplicate, using the protocol described in the
Detailed Protocol. Incubation times or temperatures significantly different from those specified may
give erroneous results.
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2. Poor duplicates, accompanied by elevated values for wells containing no sample, indicate insufficient
washing. If all instructions in the Detailed Protocol were followed accurately, such results indicate a
need for washer maintenance.
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3. Overall low signal may indicate that desiccation of the plate has occurred between the final wash and
addition of Substrate Reagent. Do not allow the plate to dry out. Add Substrate Reagent immediately
after wash.
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Reagent Stability
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Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
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All of the reagents included in the CycLex Research Product CircuLex Dog Adiponectin ELISA Kit
have been tested for stability. Reagents should not be used beyond the stated expiration date. Upon
receipt, kit reagents should be stored at 4°C, except the reconstituted Dog Adiponectin Standard must be
stored at below -70°C. Coated assay plates should be stored in the original foil bag sealed by the zip lock
and containing a desiccant pack.
For research use only, not for use in diagnostic or therapeutic procedures
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Assay Characteristics
1. Sensitivity
* Dilution Buffer is pipetted into blank wells.
Typical Standard Curve
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Twenty-one assays were evaluated and the minimum detectable dose (MDD) of dog adiponectin.
The MDD (defined as such a concentration of dog adiponectin giving absorbance higher than mean
absorbance of blank* plus three standard deviations of the absorbance of blank: A blank + 3SD blank) is
better than 163.4 pg/ml of sample.
Dog adiponectin standard curve
3 .0
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2 .5
1 .5
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A 45 0
2 .0
1 .0
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0 .5
0 .0
2 .5
5 .0
7.5
10 .0
Dog adipon ec tin c onc . ( ng/ml)
12.5
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Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
2. Precision
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Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested fourteen times on one plate to assess intra-assay
precision.
• Intra-assay (Within-Run, n=14), CV=3.1-5.1 %
Dog adiponectin conc. (ug/ml)
Serum 2
Serum 3
20.0
36.7
18.9
35.3
18.7
34.0
17.9
33.1
18.4
34.1
20.0
35.4
20.2
38.5
20.3
37.3
19.2
36.2
19.0
34.3
17.8
33.3
17.5
34.2
18.0
35.6
19.6
38.3
20.28
38.53
17.46
33.07
18.97
35.44
0.960
1.747
5.1%
4.9%
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Serum 1
13.9
14.0
14.0
14.5
14.3
14.1
14.2
13.4
13.3
13.4
13.4
13.7
14.3
14.5
14.55
13.29
13.93
0.428
3.1%
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Serum No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Max.
Min.
Mean
S.D.
C.V.
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in four separate assays to assess inter-assay
precision.
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• Inter-assay (Run-to-Run, n=4), CV=6.0-7.1 %
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1
2
3
4
Max.
Min.
Mean
S.D.
C.V.
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Cat#: CY-8052
Dog adiponectin conc. (ug/ml)
Serum 1
14.0
14.4
14.0
12.2
14.4
12.2
13.7
1.0
7.1%
Serum 2
19.4
21.3
19.9
18.4
21.3
18.4
19.7
1.2
6.0%
12
Serum 3
35.3
40.0
36.1
39.5
40.0
35.3
37.7
2.3
6.2%
Version#: 150119
Dog Adiponectin ELISA Kit
User’s Manual
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3. Linearity
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of
Adiponectin were serially diluted with the Dilution Buffer to produce samples with values within the
dynamic range of the assay.
Linearity
10.0
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▲ Se rum 2
◆ Se rum 3
7.5
5.0
2.5
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Dog adiponectin conc. (ng/ml)
● Se rum 1
0.0
0
0.5
1
1.5
2
2.5
Serum Dilution Ratio (1/10,000)
Example of Test Results
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Fig.1 Concentrations of adiponectin in female dog sera (n = 72) and male dog sera (n = 83)
Dog serum adiponectin
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50
40
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Dog adipon ectin co nc. (ug/ml)
60
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20
10
0
Male
Female
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Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
TM
Fig.2 Relationship between age and serum adiponectin concentration in female dog sera (n = 72) and
male dog sera (n = 83)
Re l at io n sh i p be t we e n Age an d Adi po n e c t i n c o n c .
< Fe m ale >
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50
25
0
0.0
2.5
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Do g adi po n e c t in c o n c . (u g/ m l )
75
5.0
7.5
10.0
Age (ye ar )
12.5
15.0
17.5
Re lat io n sh ip be t we e n Age an d Adipo n e c t in c o n c .
< M ale >
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50
25
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Dog Adipon e c t in c o n c . (u g/ m l)
75
0
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0.0
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2.5
5.0
7.5
10.0
12.5
15.0
17.5
Age (ye ar )
14
Version#: 150119
Dog Adiponectin ELISA Kit
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
References
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1. Maeda, K. et al.(1996) Biochem. Biophys. Res. Commun. 221:286.
2. Kishore, U. and K.B. Reid (2000) Immunopharmacology 49:159.
3. Shapiro, L. and P.E. Scherer (1998) Curr. Biol. 8:335.
4. Nakano, Y. et al.(1996) J. Biochem. (Tokyo) 120:803.
5. Scherer, P.E. et al. (1995) J. Biol. Chem. 270:26746.
6. Fruebis, J. et al. (2001) Proc. Natl. Acad. Sci. USA 98:2005.
7. Berg, A.H. et al. (2002) Trends Endocrinol. Metab. 13:84.
8. Arita, Y. et al. (1999) Biochem. Biophys. Res. Commun. 257:79.
9. Yamauchi, T. et al.(2003) Nature 423:762.
10 Stefan, N. et al.(2002) J. Clin. Endocrinol. Metab. 87:4652.
11. Matsubara, M. et al.(2002) Eur. J. Endocrinol. 147:173.
12. .Weyer, C. et al. (2001) J. Clin. Endocrinol. Metab. 86:1930.
13. Hotta, K. et al. (2000) Arterioscler. Thromb. Vasc. Biol. 20:1595.
14. Tomas, E. et al.(2002) Proc. Natl. Acad. Sci. USA 99:16309.
15. Berg, A.H. et al.(2001) Nat. Med. 7:947.
16. Fruebis, J. et al. (2001) Proc. Natl. Acad. Sci. USA 98:2005.
17. Yamauchi, T. et al.(2001) Nat. Med. 7:941.
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Related Products
PRODUCED BY
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CycLex Co., Ltd.
1063-103 Terasawaoka
Ina, Nagano 396-0002
Japan
Fax: +81-265-76-7618
e-mail: [email protected]
URL: http://www.cyclex.co.jp
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* CircuLex Rat Adiponectin ELISA Kit: Cat# CY-8049
* CircuLex Human Adiponectin ELISA Kit: Cat# CY-8050
* CircuLex Mouse Adiponectin ELISA Kit: Cat# CY-8051
* CircuLex Dog Adiponectin ELISA Kit: Cat# CY-8052
* Anti-Human Adiponectin: Cat# CY-P1017
* Anti-Mouse Adiponectin: Cat# CY-P1018
* Anti-Human Adiponectin (17-36 aa): Cat# CY-P1031
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CycLex/CircuLex products are supplied for research use only. CycLex/CircuLex products and
components thereof may not be resold, modified for resale, or used to manufacture commercial
products without prior written approval from CycLex Co., Ltd.. To inquire about licensing for
such commercial use, please contact us via email.
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