Human RAP1A (Ras-related protein Rap-1A) ELISA Kit (HUES02922)

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HUES02922
€539

Description

ELISA Kit Technical ManualMSDS

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.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human RAP1A in samples. No significant cross-reactivity or interference between Human RAP1A 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
(ng/mL)
O.D Average Corrected
102.467
2.527
2.4972.426
51.679
1.703
1.6911.62
2.50.997
0.959
0.9780.907
1.250.483
0.521
0.5020.431
0.630.29
0.282
0.2860.215
0.320.179
0.171
0.1750.104
0.160.124
0.124
0.1240.053
00.064
0.078
0.071--

Precision

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
Sample123123
n202020202020
Mean (ng/mL)0.521.254.490.501.224.15
Standard deviation0.030.060.180.030.070.20
C V (%)5.774.804.016.005.744.82

Recovery

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 (%)
Serum (n=5)97-108102
EDTA plasma (n=5)94-110101
Cell culture media (n=5)87-9994

Linearity

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)
1:2Range (%)93-10791-10498-113
Average (%)10098104
1:4Range (%)91-10383-9488-100
Average (%)978894
1:8Range (%)91-10282-9381-95
Average (%)968887
1:16Range (%)88-10084-9684-98
Average (%)939091

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

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]
UniProt Code:P62834
NCBI GenInfo Identifier:51338607
NCBI Gene ID:5906
NCBI Accession:P62834.1
UniProt Secondary Accession:P62834,P10113,
UniProt Related Accession:P62834
Molecular Weight:184
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
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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