Human GLUT4 (Glucose Transporter 4) ELISA Kit (HUES02783)

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HUES02783
€539

Description

ELISA Kit Technical ManualMSDS

Human GLUT4 (Glucose Transporter 4) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human GLUT4 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human GLUT4 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human GLUT4, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human GLUT4. The concentration of Human GLUT4 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human GLUT4 in samples. No significant cross-reactivity or interference between Human GLUT4 and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(ng/mL)
O.D Average Corrected
202.385
2.429
2.4072.348
101.598
1.624
1.6111.552
50.983
0.967
0.9750.916
2.50.522
0.53
0.5260.467
1.250.262
0.246
0.2540.195
0.630.176
0.154
0.1650.106
0.310.111
0.113
0.1120.053
00.05
0.068
0.059--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human GLUT4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human GLUT4 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)1.073.178.741.133.129.09
Standard deviation0.050.170.480.060.140.42
C V (%)4.675.365.495.314.494.62

Recovery

The recovery of Human GLUT4 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)94-110101
EDTA plasma (n=5)87-10194
Cell culture media (n=5)94-109101

Linearity

Samples were spiked with high concentrations of Human GLUT4 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)96-11299-11288-101
Average (%)10310694
1:4Range (%)86-10282-9681-92
Average (%)938987
1:8Range (%)87-10082-9586-98
Average (%)948792
1:16Range (%)92-10481-9381-96
Average (%)988688

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human GLUT4 (Glucose Transporter 4) ELISA Kit (HUES02783) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human GLUT4 (Glucose Transporter 4) ELISA Kit (HUES02783) Protein Information

UniProt Protein Function:GLUT4: an integral membrane facilitative glucose transporter. One of 13 members of the human equilibrative glucose transport protein family. Its surface expression is regulated by insulin. Has two internalization sequences, a dileucine repeat present in the C-terminus and a FxxY motif in the amino-terminal end. Associates with an intracellular tubulo-vesicular compartment under low plasma conditions. The binding of insulin to the insulin receptor (a typosine kinase) leads to the rapid translocation of GLUT4 to the cell surface, increasing cellular glucose transport activity. Its translocation to the plasma membrane is also stimulated by exercise, but independent from insulin signaling. This insulin-independent pathway may be regulated by AMPK.
UniProt Protein Details:

Protein type:Transporter, SLC family; Transporter; Membrane protein, multi-pass; Membrane protein, integral

Chromosomal Location of Human Ortholog: 17p13

Cellular Component: multivesicular body; trans-Golgi network transport vesicle; membrane; clathrin-coated vesicle; integral to plasma membrane; perinuclear region of cytoplasm; T-tubule; plasma membrane; endomembrane system; coated pit; vesicle membrane; cytosol; lipid raft; external side of plasma membrane

Molecular Function:D-glucose transmembrane transporter activity; protein binding; glucose transmembrane transporter activity

Biological Process: amylopectin biosynthetic process; response to ethanol; cellular response to insulin stimulus; glucose import; hexose transport; carbohydrate metabolic process; brown fat cell differentiation; pathogenesis; glucose transport; glucose homeostasis; transmembrane transport

Disease: Diabetes Mellitus, Noninsulin-dependent

NCBI Summary:This gene is a member of the solute carrier family 2 (facilitated glucose transporter) family and encodes a protein that functions as an insulin-regulated facilitative glucose transporter. In the absence of insulin, this integral membrane protein is sequestered within the cells of muscle and adipose tissue. Within minutes of insulin stimulation, the protein moves to the cell surface and begins to transport glucose across the cell membrane. Mutations in this gene have been associated with noninsulin-dependent diabetes mellitus (NIDDM). [provided by RefSeq, Jul 2008]
UniProt Code:P14672
NCBI GenInfo Identifier:121761
NCBI Gene ID:6517
NCBI Accession:P14672.1
UniProt Secondary Accession:P14672,Q05BQ3, Q14CX2,
UniProt Related Accession:P14672
Molecular Weight:509
NCBI Full Name:Solute carrier family 2, facilitated glucose transporter member 4
NCBI Synonym Full Names:solute carrier family 2 (facilitated glucose transporter), member 4
NCBI Official Symbol:SLC2A4  
NCBI Official Synonym Symbols:GLUT4  
NCBI Protein Information:solute carrier family 2, facilitated glucose transporter member 4; GLUT-4; insulin-responsive glucose transporter type 4; glucose transporter type 4, insulin-responsive
UniProt Protein Name:Solute carrier family 2, facilitated glucose transporter member 4
UniProt Synonym Protein Names:Glucose transporter type 4, insulin-responsive; GLUT-4
UniProt Gene Name:SLC2A4  
UniProt Entry Name:GTR4_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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