Glucagon (GCG) ELISA Kit

Details for Product No. ABIN1979125, Supplier: Log in to see
Antigen
  • GLP1
  • GLP2
  • GRPP
  • GLP-1
  • Glu
  • PPG
  • GCG
  • gcg-A
  • gcg1
  • glucagon
  • glucagon L homeolog
  • GCG
  • Gcg
  • gcg.L
Reactivity
Human, Mouse (Murine), Rat (Rattus)
Alternatives
Kits with alternative reactivity to:
39
33
31
11
10
10
4
4
3
2
2
1
1
1
Method Type
Competition ELISA
Detection Range
1-1.000 pg/mL
Minimum Detection Limit
1 pg/mL
Application
ELISA
Options
Supplier
Log in to see
Supplier Product No.
Log in to see
Purpose Human/Mouse/Rat Glucagon EIA Kit optimized for serum and cell culture medium. Competition-based ELISA on a 96-well strip plate.
Sample Type Cell Culture Supernatant, Serum
Analytical Method Quantitative
Detection Method Colorimetric
Specificity Cross Reactivity: This EIA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, Angiotensin II, NPY and APC
Cross-Reactivity (Details) This ELISA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, Angiotensin II, NPY and APC
Sensitivity 4.77 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
  • Standard Peptide
  • Assay Diluent(s)
  • Biotinylated Peptide
  • HRP-Streptavidin
  • TMB One-Step Substrate
  • Stop Solution
  • Assay Diagram
  • Positive Control Sample
  • Capture Antibody
  • User Manual
Material not included
  • Distilled or deionized water
  • Precision pipettes to deliver 2 μL to 1 mL volumes
  • Adjustable 1-25 mL pipettes for reagent preparation
  • 100 mL and 1 liter graduated cylinders
  • Tubes to prepare standard and sample dilutions
  • Orbital shaker
  • Aluminum foil
  • Saran Wrap
  • Absorbent paper
  • Microplate reader capable of measuring absorbance at 450nm
  • SigmaPlot software (or other software that can perform four-parameter logistic regression models)
Alternative Name Glucagon (GCG ELISA Kit Abstract)
Background Glucagon is a 29-amino acid peptide hormone secreted by the pancreas. Its function is to raise blood glucose levels to opposing the effect of insulin, which lowers blood glucose levels. Glucagon is synthesized and secreted from alpha cells of the endocrine portion of the pancreas. In rodents, the alpha cells are located in the outer rim of the islet. However, alpha cells in human pancreas are distributed throughout the islet. Glucagon and insulin are part of a feedback system that keeps blood glucose levels at a stable level. The pancreas releases glucagon when glucose levels fall too low. Glucagon causes the liver to convert stored glycogen into glucose, which is released into the bloodstream. Glucagon raises blood glucose levels. High blood glucose levels stimulate the release of insulin. Insulin allows glucose to be taken up and used by insulin-dependent tissues. Glucagon has important clinical applications. Abnormally-elevated levels of glucagon may be caused by pancreatic tumors, such as glucagonoma, with symptoms including necrolytic migratory erythema, reduced amino acids, and hyperglycemia. It may occur alone or in the context of multiple endocrine neoplasia type 1 (MENI).
Gene ID 14526
UniProt P55095
Pathways Positive Regulation of Peptide Hormone Secretion, Peptide Hormone Metabolism, cAMP Metabolic Process, Regulation of Carbohydrate Metabolic Process, Feeding Behaviour, Negative Regulation of intrinsic apoptotic Signaling
Application Notes Recommended Dilution for serum and plasma samplesHuman: 2X / Mouse: 2X / Rat: 2X
Sample Volume 100 μL
Assay Time 5 h
Plate Pre-coated
Protocol
  1. Prepare all reagents, samples and standards as instructed.
  2. Add 100 μL detection antibody to each well.
  3. Incubate 1.5 h at RT or O/N at 4 °C.
  4. Add 100 μL standard or sample to each well.
  5. Incubate 2.5 h at RT.
  6. Add 100 μL prepared streptavidin solution.
  7. Incubate 45 min at RT.
  8. Add 100 μL TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 μL Stop Solution to each well.
  11. Read plate at 450 nm immediately.
Reagent Preparation
  1. Keep kit reagents on ice during steps. Equilibrate plate to room temperature before opening the sealed pouch.
    2. Briefly centrifuge the Glucagon Antibody vial (Item N) and reconstitute with 5 µL of ddH2O before use. Add 50 µL of 1x Assay Diluent E into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently.
    3. The antibody concentrate should then be diluted 100-fold with 1x Assay Diluent E. This is your anti-Glucagon antibody working solution, which will be used in step 2 of the Assay Procedure. NOTE: the following steps may be done during the antibody incubation procedure (step 2 of Assay Procedure).
    4. Briefly centrifuge the vial of biotinylated Glucagon peptide (Item F) and reconstitute with 20 µL of ddH2O before use. Add 5 µL of Item F to 5 mL 1X Assay Diluent E. Pipette up and down to mix gently. The final concentration of biotinylated Glucagon will be 40 pg/mL. This solution will only be used as the diluent in step 5 of Reagent Preparation.
    5. Preparation of Standards: Label 6 microtubes with the following concentrations: 1000 pg/mL, 100 pg/mL, 10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0 pg/mL. Pipette 450 mL of biotinylated Glucagon solution into each tube, except for the 1000 pg/mL (leave this one empty). It is very important to make sure the concentration of biotinylated Glucagon is 40 pg/mL in all standards. a. Briefly centrifuge the vial of standard Glucagon peptide (Item C) and reconstitute with 10 µL of ddH2O. In the tube labeled 1000 pg/mL, pipette 8 µL of Item C and 792 µL of 40 pg/mL biotinylated Glucagon solution (prepared in step 4 above). This is your Glucagon stock solution (1000 pg/mL Glucagon, 40 pg/mL biotinylated Glucagon). Mix thoroughly. This solution serves as the first standard. b. To make the 100 pg/mL standard, pipette 50 µL of Glucagon stock solution the tube labeled 100 pg/mL. Mix thoroughly. c. Repeat this step with each successive concentration, preparing a dilution series as shown in the illustration below. Each time, use 450 mL of biotinylated Glucagon and 50 mL of the prior concentration until 0.1 pg/mL is reached. Mix each tube thoroughly before the next transfer. d. The final tube (0 pg/mL Glucagon, 40 pg/mL biotinylated Glucagon) serves as the zero standard (or total binding).
    6. Prepare a 10-fold dilution of Item F. To do this, add 2 mL of Item F to 18 mL of 1X Assay Diluent E. This solution will be used in steps 7 and
    9.
    7. Positive Control Preparation: Briefly centrifuge the positive control vial and reconstitute with 100 µL of ddH2O before use (Item M). To the tube of Item M, add 101 µL 1x Assay Diluent E. Also add 2 µL of 10-fold diluted Item F (prepared in step 6) to the tube. This is a 2-fold dilution of the positive control. Mix thoroughly. The positive control is a cell culture medium sample that is meant to be a system control (to verify that the detection & kit components are working). It may be diluted further if desired, but be sure the final concentration of biotinylated Glucagon is 40 pg/mL.
    8. If Item B (20X Wash Concentrate) 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. 1000 100 10 1 0.1 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL 50 mL 50 mL 50 mL 50 mL
    9. Sample Preparation: Use 1X Assay Diluent E + biotinylated Glucagon to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Glucagon is 40 pg/mL in every sample.
    Example: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 mL of 1X Assay Diluent E, and 62.5 mL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Glucagon to a final concentration of 40 pg/mL.
    Example: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.
    10. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. The HRP-Streptavidin concentrate should be diluted 200- fold with 1X Assay Diluent E.
Sample Preparation

Use 1X Assay Diluent E + biotinylated Glucagon to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Glucagon is 40 pg/mL in every sample. EXAMPLE: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 mL of 1X Assay Diluent E, and 62.5 mL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Glucagon to a final concentration of 40 pg/mL. EXAMPLE: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.

Assay Procedure
  1. Keep kit reagents on ice during reagent preparation steps. It is recommended that all standards and samples be run at least in duplicate.
    2. Add 100 µL anti-Glucagon antibody (see Reagent Preparation step 3) to each well. Incubate for 1.5 hours at room temperature with gentle shaking (1-2 cycles/sec). You may also incubate overnight at 4 degrees C. 0
    3. Discard the solution and wash wells 4 times with 1x Wash Buffer (200-300 µL each). Washing may be done with a multichannel pipette or an automated plate washer. Complete removal of liquid at each step is essential to good assay 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 each standard (see Reagent Preparation step 5), positive control (see Reagent Preparation step 7) and sample (see Reagent Preparation step 9) into appropriate wells. Be sure to include a blank well (Assay Diluent only). Cover wells and incubate for 2.5 hours at room temperature with gentle shaking (1-2 cycles/sec) or overnight at 4 °C.
    5. Discard the solution and wash 4 times as directed in Step
    3.
    6. Add 100 µL of prepared HRP-Streptavidin solution (see Reagent Preparation step 10) to each well. Incubate for 45 minutes with gentle shaking at room temperature. It is recommended that incubation time should not be shorter or longer than 45 minutes.
    7. Discard the solution and wash 4 times as directed in Step
    3.
    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 (1-2 cycles/sec).
    9. Add 50 µL of Stop Solution (Item I) to each well. Read absorbances at 450 nm immediately. 1
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the blank optical density. Plot the standard curve using SigmaPlot software (or other software which can perform four-parameter logistic regression models), with standard concentration on the x-axis and percentage of absorbance on the y-axis. Draw the best-fit curve through the standard points.

Assay Precision Intra-Assay: CV < 10 %
Inter-Assay: CV < 15 %
Restrictions For Research Use only
Handling Advice Avoid repeated freeze/thaw cycles.
Storage -20 °C
Storage Comment Standard, Biotinylated Glucagon peptide, and Positive Control should be stored at -20°C after arrival. Avoid multiple freeze-thaws. The remaining kit components may be stored at 4°C. Opened Microplate Wells and antibody (Item N) may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.
Expiry Date 6 months
Did you look for something else?