Rat GDN(Glia Derived Nexin)ELISA Kit  (RTES01148)

SKU:
RTES01148
Product type:
ELISA
Reactivity:
Rat
ELISA Type:
Sandwich
€649

Description

ELISA Kit Technical ManualMSDS

Rat GDN(Glia Derived Nexin)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 GDN . 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 GDN 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 GDN, 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 GDN. The concentration of Rat GDN 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 Range6.25-400 pg/mL
Sensitivity3.75 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Rat GDN in samples. No significant cross-reactivity or interference between Rat GDN 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
4002.524
2.544
2.5342.455
2001.646
1.694
1.671.591
1000.907
0.901
0.9040.825
500.441
0.455
0.4480.369
250.253
0.235
0.2440.165
12.50.192
0.17
0.1810.102
6.250.123
0.139
0.1310.052
00.079
0.079
0.079--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)20.5254.62151.1019.2451.86165.87
Standard deviation1.113.017.711.292.515.18
C V (%)5.415.515.106.704.843.12

Recovery

The recovery of Rat GDN 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)86-10091
EDTA plasma (n=5)85-9991
Cell culture media (n=5)94-10699

Linearity

Samples were spiked with high concentrations of Rat GDN 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 (%)96-11293-10791-106
Average (%)1039897
1:4Range (%)91-10382-9586-96
Average (%)978891
1:8Range (%)91-10680-9386-98
Average (%)988692
1:16Range (%)90-10286-9786-99
Average (%)969193

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 GDN(Glia Derived Nexin)ELISA Kit  (RTES01148) 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 GDN(Glia Derived Nexin)ELISA Kit  (RTES01148) Protein Information

UniProt Protein Function:SERPINE2: Serine protease inhibitor with activity toward thrombin, trypsin, and urokinase. Promotes neurite extension by inhibiting thrombin. Binds heparin. Belongs to the serpin family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Secreted; Inhibitor; Secreted, signal peptide

Cellular Component: extracellular matrix; extracellular space; extrinsic to external side of plasma membrane; membrane; cell soma; cell; extracellular region; cytosol; neuromuscular junction; vesicle

Molecular Function:serine-type endopeptidase inhibitor activity; heparin binding; glycosaminoglycan binding; receptor binding

Biological Process: negative regulation of proteolysis; regulation of synaptic transmission, glutamatergic; secretory granule organization and biogenesis; cerebellar granular layer morphogenesis; negative regulation of peptidase activity; negative regulation of blood coagulation; detection of mechanical stimulus involved in sensory perception; mating plug formation; negative regulation of cell proliferation; response to radiation; negative regulation of smoothened signaling pathway; response to wounding; negative regulation of cell growth; blood coagulation; negative regulation of protein catabolic process; regulation of timing of cell differentiation; embryo implantation; negative regulation of phosphoinositide 3-kinase cascade; secretion by cell; positive regulation of astrocyte differentiation

NCBI Summary:displays neurite promoting activity and serine protease inhibitory activity [RGD, Feb 2006]
UniProt Code:P07092
NCBI GenInfo Identifier:158138559
NCBI Gene ID:29366
NCBI Accession:NP_062070.1
UniProt Related Accession:P07092
Molecular Weight:44,063 Da
NCBI Full Name:glia-derived nexin
NCBI Synonym Full Names:serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2
NCBI Official Symbol:Serpine2  
NCBI Official Synonym Symbols:PI-7; Pn-1; Spin4; Gdnpn1  
NCBI Protein Information:glia-derived nexin; GDN; serpin E2; protease nexin 1; protease nexin I; protease nexin PN-1; peptidase inhibitor 7; serine protease inhibitor 4; Glia-derived nexin/protease nexin I; plasminogen activator inhibitor type 1; serpin peptidase inhibitor, clade E, member 2; serine (or cysteine) peptidase inhibitor, clade E, member 2; serine (or cysteine) proteinase inhibitor, clade E, member 2
UniProt Protein Name:Glia-derived nexin
UniProt Synonym Protein Names:Peptidase inhibitor 7; PI-7; Protease nexin 1; PN-1; Protease nexin I; Serpin E2
Protein Family:Glia-derived nexin
UniProt Gene Name:Serpine2  
UniProt Entry Name:GDN_RAT
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