Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	0.16-10 ng/mL
Sensitivity:	0.10 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse GAPDH in samples. No significant cross-reactivity or interference between Mouse GAPDH and analogues was observed.

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 Mouse GAPDH. 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 Mouse GAPDH 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 Mouse GAPDH, 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 Mouse GAPDH. The concentration of Mouse GAPDH in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	GAPDH: a multifunctional enzyme with both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities. A key glycolytic enzyme that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate. An important enzyme for energy metabolism, and the production of ATP and pyruvate through anaerobic glycolysis in the cytoplasm. Additionally, it participates in apoptosis, membrane trafficking, iron metabolism, nuclear activities and receptor mediated cell signaling. Its subcellular localization changes reflecting its multiple activities. Is cytosolic, but is also localized in the membrane, the nucleus, polysomes, the ER and the Golgi. Participates in transcription, RNA transport, DNA replication and apoptosis. S-nitrosylation on Cys-152 following apoptotic stimulates its interaction with SIAH2, which in turn moderates its translocation into the nucleus. Mediates cysteine S-nitrosylation of nuclear target proteins including SIRT1, HDAC2 and DNA-PK. Deregulated in lung cancer, renal cancer, breast cancer, gastric cancer, glioma, liver cancer, colorectal cancer, melanoma, prostatic cancer, pancreatic cancer and bladder cancer. Its increased expression and enzymatic activity is associated with cell proliferation and tumorigenesis, Oxidative stress impairs GAPDH catalytic activity and leads to cellular aging and apoptosis. In experimental animal models, injection of GAPDH antagonists induces apoptosis and blocks Hep3B tumor progression, suggesting a therapeutic potential of targeting GAPDH in human hepatocellular carcinoma
UniProt Protein Details:	Protein type:EC 1. 2. 1. 12; Oxidoreductase; Carbohydrate Metabolism - glycolysis and gluconeogenesis Cellular Component: cytoplasm; cytosol; intracellular membrane-bound organelle; lipid particle; membrane; microtubule cytoskeleton; mitochondrion; nuclear membrane; nucleus; plasma membrane; ribonucleoprotein complex; vesicle Molecular Function:glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) activity; identical protein binding; microtubule binding Biological Process: apoptosis; carbohydrate metabolic process; gluconeogenesis; glycolysis; microtubule cytoskeleton organization and biogenesis; multicellular organismal development; negative regulation of translation; neuron apoptosis; protein stabilization
UniProt Code:	P16858
NCBI GenInfo Identifier:	120702
NCBI Gene ID:	100042025
NCBI Accession:	P16858. 2
UniProt Secondary Accession:	P16858,Q0QEU0, Q3THM2, Q3TUI2, Q3UMT2, Q4V783, Q569X2 Q569X5, Q5U410, A6H6A8,
Molecular Weight:	35,810 Da
NCBI Full Name:	Glyceraldehyde-3-phosphate dehydrogenase
NCBI Synonym Full Names:	glyceraldehyde-3-phosphate dehydrogenase, pseudogene 15
NCBI Official Symbol:	Gapdh-ps15
NCBI Official Synonym Symbols:	Gm20899
NCBI Protein Information:	glyceraldehyde-3-phosphate dehydrogenase, pseudogene 15
UniProt Protein Name:	Glyceraldehyde-3-phosphate dehydrogenase
UniProt Synonym Protein Names:	Peptidyl-cysteine S-nitrosylase GAPDH (EC:2. 6. 99. -)
UniProt Gene Name:	Gapdh
UniProt Entry Name:	G3P_MOUSE

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
10	2.384 2.388	2.386	2.298
5	1.511 1.539	1.525	1.437
2.5	0.854 0.854	0.854	0.766
1.25	0.421 0.441	0.431	0.343
0.63	0.27 0.254	0.262	0.174
0.32	0.184 0.18	0.182	0.094
0.16	0.128 0.146	0.137	0.049
0	0.083 0.093	0.088	--

Precision

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

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

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.53	1.22	4.48	0.58	1.10	4.40
Standard deviation	0.03	0.06	0.15	0.04	0.06	0.15
C V (%)	5.66	4.92	3.35	6.90	5.45	3.41

Recovery

The recovery of Mouse GAPDH 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)	87-102	93
EDTA plasma (n=5)	90-103	96
Cell culture media (n=5)	84-96	90

Linearity

Samples were spiked with high concentrations of Mouse GAPDH 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)
1:2	Range (%)	88-100	97-112	98-115
1:2	Average (%)	95	102	105
1:4	Range (%)	89-99	82-98	83-94
1:4	Average (%)	94	89	88
1:8	Range (%)	86-102	81-94	81-95
1:8	Average (%)	93	86	88
1:16	Range (%)	91-107	82-94	87-100
1:16	Average (%)	98	87	92

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 ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

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.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
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.
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.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
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.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.