Human RAP1A (Ras-related protein Rap-1A) ELISA Kit (HUES02922)
Human RAP1A (Ras-related protein Rap-1A) 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 Human RAP1A . 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 RAP1A 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 RAP1A, 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 RAP1A. The concentration of Human RAP1A in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||0.16-10 ng/mL|
|Sample Volume Required Per Well||100uL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Human RAP1A in samples. No significant cross-reactivity or interference between Human RAP1A and analogues was observed.
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 RAP1A were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human RAP1A were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.77||4.80||4.01||6.00||5.74||4.82|
The recovery of Human RAP1A 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)||94-110||101|
|Cell culture media (n=5)||87-99||94|
Samples were spiked with high concentrations of Human RAP1A 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)|
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.
|Micro ELISA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 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 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 (25X)||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|
Human RAP1A (Ras-related protein Rap-1A) ELISA Kit (HUES02922) 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.
Human RAP1A (Ras-related protein Rap-1A) ELISA Kit (HUES02922) Protein Information
|UniProt Protein Function:||RAP1A: a small G protein of the Ras family with tumor suppressor activity. Induces morphological reversion of a cell line transformed by a Ras oncogene. Activated by guanine nucleotide-exchange factors (GEF) EPAC and EPAC2 in a cAMP-dependent manner, and GFR. Counteracts the mitogenic function of Ras, at least partly because it can interact with Ras GAPs and RAF in a competitive manner. Interacts with SGSM1, SGSM2 and SGSM3.|
|UniProt Protein Details:|
Protein type:Tumor suppressor; G protein, monomeric; Motility/polarity/chemotaxis; G protein; G protein, monomeric, Ras
Chromosomal Location of Human Ortholog: 1p13.3
Cellular Component: neuron projection; perinuclear region of cytoplasm; late endosome; cytoplasm; early endosome; plasma membrane; cell junction; cytosol
Molecular Function:GTPase activity; Rap guanyl-nucleotide exchange factor activity; protein binding; Ras GTPase binding; GTP binding; protein complex binding; protein transporter activity
Biological Process: platelet activation; microvillus biogenesis; protein transport; nerve growth factor receptor signaling pathway; activation of MAPKK activity; positive regulation of protein kinase activity; energy reserve metabolic process; blood coagulation; signal transduction; regulation of insulin secretion; Rap protein signal transduction
|NCBI Summary:||This gene encodes a member of the Ras family of small GTPases. The encoded protein undergoes a change in conformational state and activity, depending on whether it is bound to GTP or GDP. This protein is activated by several types of guanine nucleotide exchange factors (GEFs), and inactivated by two groups of GTPase-activating proteins (GAPs). The activation status of the encoded protein is therefore affected by the balance of intracellular levels of GEFs and GAPs. The encoded protein regulates signaling pathways that affect cell proliferation and adhesion, and may play a role in tumor malignancy. Pseudogenes of this gene have been defined on chromosomes 14 and 17. Alternative splicing results in multiple transcript variants. [provided by RefSeq, May 2014]|
|NCBI GenInfo Identifier:||51338607|
|NCBI Gene ID:||5906|
|UniProt Secondary Accession:||P62834,P10113,|
|UniProt Related Accession:||P62834|
|NCBI Full Name:||Ras-related protein Rap-1A|
|NCBI Synonym Full Names:||RAP1A, member of RAS oncogene family|
|NCBI Official Symbol:||RAP1A|
|NCBI Official Synonym Symbols:||RAP1; C21KG; G-22K; KREV1; KREV-1; SMGP21|
|NCBI Protein Information:||ras-related protein Rap-1A; Ras-related protein Krev-1; GTP-binding protein smg p21A|
|UniProt Protein Name:||Ras-related protein Rap-1A|
|UniProt Synonym Protein Names:||C21KG; G-22K; GTP-binding protein smg p21A; Ras-related protein Krev-1|
|Protein Family:||Ras-related protein|
|UniProt Gene Name:||RAP1A|
|UniProt Entry Name:||RAP1A_HUMAN|