Human GAP (GTPase Activating Protein) ELISA Kit (HUES02013)
- Product Type:
- ELISA Kit
- 96 Assays
- ELISA Type:
- Tested Sample Types:
- Serum, plasma and other biological fluids
|Detection Range:||0.16-10 ng/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Human GAP in samples. No significant cross-reactivity or interference between Human GAP 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 Human GAP. 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 GAP 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 GAP, 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 GAP. The concentration of Human GAP in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||RASA1: a GTPase activator for normal RAS p21 but not its oncogenic counterpart, converting it to the putatively inactive GDP-bound state. Acting as a suppressor of RAS function, it enhances the weak intrinsic GTPase activity of RAS proteins resulting in the inactive GDP-bound form of RAS, thereby allowing control of cellular proliferation and differentiation. Mutations leading to changes in the binding sites of either protein are associated with basal cell carcinomas. Two alternatively spliced isoforms have been described.|
|UniProt Protein Details:|
Protein type:Motility/polarity/chemotaxis; GAPs, Ras; Oncoprotein; GAPs
Chromosomal Location of Human Ortholog: 5q13. 3
Cellular Component: ruffle; cytoplasm; cytosol
Molecular Function:GTPase binding; protein binding; glycoprotein binding; potassium channel inhibitor activity; receptor binding
Biological Process: axon guidance; negative regulation of cell adhesion; negative regulation of cell-matrix adhesion; signal transduction; regulation of cell shape; regulation of actin filament polymerization; embryonic development; cytokinesis after mitosis; regulation of RNA metabolic process; ephrin receptor signaling pathway; blood vessel morphogenesis; negative regulation of Ras protein signal transduction; negative regulation of neuron apoptosis; vascular endothelial growth factor receptor signaling pathway; vasculogenesis
Disease: Parkes Weber Syndrome; Capillary Malformation-arteriovenous Malformation; Basal Cell Carcinoma, Susceptibility To, 1
|NCBI Summary:||The protein encoded by this gene is located in the cytoplasm and is part of the GAP1 family of GTPase-activating proteins. The gene product stimulates the GTPase activity of normal RAS p21 but not its oncogenic counterpart. Acting as a suppressor of RAS function, the protein enhances the weak intrinsic GTPase activity of RAS proteins resulting in the inactive GDP-bound form of RAS, thereby allowing control of cellular proliferation and differentiation. Mutations leading to changes in the binding sites of either protein are associated with basal cell carcinomas. Mutations also have been associated with hereditary capillary malformations (CM) with or without arteriovenous malformations (AVM) and Parkes Weber syndrome. Alternative splicing results in two isoforms where the shorter isoform, lacking the N-terminal hydrophobic region but retaining the same activity, appears to be abundantly expressed in placental but not adult tissues. [provided by RefSeq, May 2012]|
|NCBI GenInfo Identifier:||4506431|
|NCBI Gene ID:||5921|
|NCBI Accession:||NP_002881. 1|
|UniProt Secondary Accession:||P20936,Q68CU6, Q9UDI1, B2R6W3, B4DTL2,|
|UniProt Related Accession:||P20936|
|Molecular Weight:||101,546 Da|
|NCBI Full Name:||ras GTPase-activating protein 1 isoform 1|
|NCBI Synonym Full Names:||RAS p21 protein activator 1|
|NCBI Official Symbol:||RASA1|
|NCBI Official Synonym Symbols:||GAP; PKWS; RASA; p120; CMAVM; CM-AVM; RASGAP; p120GAP; p120RASGAP|
|NCBI Protein Information:||ras GTPase-activating protein 1|
|UniProt Protein Name:||Ras GTPase-activating protein 1|
|UniProt Synonym Protein Names:||Ras p21 protein activator; p120GAP|
|Protein Family:||Ras GTPase-activating protein|
|UniProt Gene Name:||RASA1|
|UniProt Entry Name:||RASA1_HUMAN|
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human GAP were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human GAP were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.45||3.96||4.07||7.02||5.45||3.68|
The recovery of Human GAP spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||84-96||90|
|Cell culture media (n=5)||86-99||91|
Samples were spiked with high concentrations of Human GAP 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)|
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.
|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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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.