Rat GLUT4 (Glucose Transporter 4)CLIA Kit (RTES00246)

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RTES00246
€649

Description

ELISA Kit Technical ManualMSDS

Rat GLUT4 (Glucose Transporter 4)CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Rat Glut4 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat 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 Rat Glut4, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Rat Glut4. The concentration of Rat Glut4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection methodChemiluminescence
Detection range125.00-8000 pg/mL
Sensitivity75.00 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

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

Typical Data

As the RLU 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
(pg/mL)
RLU Average Corrected
800065448
77430
7143971418
400032568
38428
3549835477
200018199
16909
1755417533
10008384
8792
85888567
5004108
4106
41074086
2501889
1845
18671846
125.00721
773
747726
021
21
21--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)405.19808.133367.24430.94727.813612.82
Standard deviation52.3195.93292.2845.3885.59305.28
C V (%)12.9111.878.6810.5311.768.45

Recovery

The recovery of Rat 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)99-115106
EDTA plasma (n=5)96-111101
Cell culture media (n=5)86-9792

Linearity

Samples were spiked with high concentrations of Rat 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 (%)96-11197-108102-115
Average (%)102102108
1:4Range (%)100-11983-9590-104
Average (%)1099095
1:8Range (%)99-11485-97102-118
Average (%)10691108
1:16Range (%)92-10587-100101-114
Average (%)9893108

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 CLIA 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 Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Rat GLUT4 (Glucose Transporter 4)CLIA Kit (RTES00246) 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Rat GLUT4 (Glucose Transporter 4)CLIA Kit (RTES00246) 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; Membrane protein, multi-pass; Transporter; Membrane protein, integral

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

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

Biological Process: amylopectin biosynthetic process; brown fat cell differentiation; cellular response to insulin stimulus; glucose homeostasis; glucose transport; response to ethanol

NCBI Summary:transports glucose; may play a role in insulin response [RGD, Feb 2006]
UniProt Code:P19357
NCBI GenInfo Identifier:121763
NCBI Gene ID:25139
NCBI Accession:P19357.1
UniProt Secondary Accession:P19357,P97900,
UniProt Related Accession:P19357
Molecular Weight:54,896 Da
NCBI Full Name:Solute carrier family 2, facilitated glucose transporter member 4
NCBI Synonym Full Names:solute carrier family 2 member 4
NCBI Official Symbol:Slc2a4  
NCBI Official Synonym Symbols:Glut4  
NCBI Protein Information:solute carrier family 2, facilitated glucose transporter member 4
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_RAT
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Additional Information

Reactivity:
Rat
ELISA Type:
CLIA
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