Rat GYS2 (Glycogen Synthase 2, Liver) ELISA Kit (RTES01084)

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RTES01084
€649

Description

ELISA Kit Technical ManualMSDS

Rat GYS2 (Glycogen Synthase 2, Liver) 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 Rat GYS2 . 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 Rat GYS2 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 GYS2, 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 Rat GYS2. The concentration of Rat GYS2 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection MethodColormetric
Detection Range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Rat GYS2 in samples. No significant cross-reactivity or interference between Rat GYS2 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
(pg/mL)
O.D Average Corrected
50002.439
2.449
2.4442.376
25001.673
1.691
1.6821.614
12500.974
0.972
0.9730.905
6250.447
0.481
0.4640.396
312.50.261
0.247
0.2540.186
156.250.178
0.176
0.1770.109
78.130.121
0.127
0.1240.056
00.066
0.07
0.068--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat GYS2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat GYS2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)233.47504.521916.68250.73519.002027.40
Standard deviation12.3323.9667.0815.1223.36107.45
C V (%)5.284.753.506.034.505.30

Recovery

The recovery of Rat GYS2 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)93-107101
EDTA plasma (n=5)83-9590
Cell culture media (n=5)84-9590

Linearity

Samples were spiked with high concentrations of Rat GYS2 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 (%)95-11090-10194-105
Average (%)1029699
1:4Range (%)89-10585-9888-100
Average (%)969193
1:8Range (%)89-10280-9183-94
Average (%)978689
1:16Range (%)91-10584-9588-99
Average (%)988994

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

Rat GYS2 (Glycogen Synthase 2, Liver) ELISA Kit (RTES01084) 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.

Rat GYS2 (Glycogen Synthase 2, Liver) ELISA Kit (RTES01084) Protein Information

UniProt Protein Function:GYS2: Transfers the glycosyl residue from UDP-Glc to the non- reducing end of alpha-1,4-glucan. Defects in GYS2 are the cause of glycogen storage disease type 0 (GSD0); A metabolic disorder characterized by fasting hypoglycemia presenting in infancy or early childhood, high blood ketones and low alanine and lactate concentrations. Although feeding relieves symptoms, it often results in postprandial hyperglycemia and hyperlactatemia. Belongs to the glycosyltransferase 3 family.
UniProt Protein Details:

Protein type:Carbohydrate Metabolism - starch and sucrose; Cytoskeletal; EC 2.4.1.11; Transferase

Chromosomal Location of Human Ortholog: 4q44

Cellular Component: cell cortex; cortical actin cytoskeleton; cytoplasm; cytoskeleton; cytosol; ectoplasm

Molecular Function:glucose binding; glycogen (starch) synthase activity

Biological Process: glycogen biosynthetic process; glycogen metabolic process; response to glucose stimulus

NCBI Summary:catalyzes the formation of 1,4-a-linked glucose chains in glycogen, the primary storage form of glucose [RGD, Feb 2006]
UniProt Code:P17625
NCBI GenInfo Identifier:218512064
NCBI Gene ID:25623
NCBI Accession:P17625.2
UniProt Secondary Accession:P17625,Q99MF8,
UniProt Related Accession:P17625
Molecular Weight:80,734 Da
NCBI Full Name:Glycogen
NCBI Synonym Full Names:glycogen synthase 2
NCBI Official Symbol:Gys2  
NCBI Official Synonym Symbols:GLYSN  
NCBI Protein Information:glycogen [starch] synthase, liver
UniProt Protein Name:Glycogen [starch] synthase, liver
Protein Family:Glycogen [starch] synthase
UniProt Gene Name:Gys2  
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
Sandwich
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