Human RAG-2 (Recombination Activating Gene 2) ELISA Kit (HUES03195)

SKU:
HUES03195
€539

Description

ELISA Kit Technical ManualMSDS

Human RAG-2 (Recombination Activating Gene 2) 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 RAG-2 . 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 RAG-2 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 RAG-2, 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 RAG-2. The concentration of Human RAG-2 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 Range15.63-1000 pg/mL
Sensitivity9.38 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human RAG-2 in samples. No significant cross-reactivity or interference between Human RAG-2 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
10002.471
2.479
2.4752.406
5001.634
1.66
1.6471.578
2501.016
0.984
10.931
1250.433
0.463
0.4480.379
62.50.242
0.234
0.2380.169
31.250.175
0.169
0.1720.103
15.630.117
0.127
0.1220.053
00.065
0.073
0.069--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human RAG-2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human RAG-2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)55.09106.58470.5158.33101.13451.94
Standard deviation2.925.9921.413.354.7920.70
C V (%)5.305.624.555.744.744.58

Recovery

The recovery of Human RAG-2 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)93-111101
EDTA plasma (n=5)88-9893
Cell culture media (n=5)85-9690

Linearity

Samples were spiked with high concentrations of Human RAG-2 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 (%)90-10492-10790-102
Average (%)959896
1:4Range (%)92-10482-9587-98
Average (%)978793
1:8Range (%)89-10082-9587-101
Average (%)948992
1:16Range (%)94-10581-9687-100
Average (%)998894

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 RAG-2 (Recombination Activating Gene 2) ELISA Kit (HUES03195) 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 RAG-2 (Recombination Activating Gene 2) ELISA Kit (HUES03195) Protein Information

UniProt Protein Function:RAG2: Core component of the RAG complex, a multiprotein complex that mediates the DNA cleavage phase during V(D)J recombination. V(D)J recombination assembles a diverse repertoire of immunoglobulin and T-cell receptor genes in developing B and T- lymphocytes through rearrangement of different V (variable), in some cases D (diversity), and J (joining) gene segments. DNA cleavage by the RAG complex occurs in 2 steps: a first nick is introduced in the top strand immediately upstream of the heptamer, generating a 3'-hydroxyl group that can attack the phosphodiester bond on the opposite strand in a direct transesterification reaction, thereby creating 4 DNA ends: 2 hairpin coding ends and 2 blunt, 5'-phosphorylated ends. The chromatin structure plays an essential role in the V(D)J recombination reactions and the presence of histone H3 trimethylated at 'Lys-4' (H3K4me3) stimulates both the nicking and haipinning steps. The RAG complex also plays a role in pre-B cell allelic exclusion, a process leading to expression of a single immunoglobulin heavy chain allele to enforce clonality and monospecific recognition by the B- cell antigen receptor (BCR) expressed on individual B-lymphocytes. The introduction of DNA breaks by the RAG complex on one immunoglobulin allele induces ATM-dependent repositioning of the other allele to pericentromeric heterochromatin, preventing accessibility to the RAG complex and recombination of the second allele. In the RAG complex, RAG2 is not the catalytic component but is required for all known catalytic activities mediated by RAG1. It probably acts as a sensor of chromatin state that recruits the RAG complex to H3K4me3. Component of the RAG complex composed of core components RAG1 and RAG2, and associated component HMGB1 or HMGB2. Cells of the B- and T-lymphocyte lineages. Belongs to the RAG2 family.
UniProt Protein Details:

Protein type:Enzyme, misc.

Chromosomal Location of Human Ortholog: 11p13

Cellular Component: nucleoplasm

Molecular Function:chromatin binding; methylated histone residue binding; phosphatidylinositol-3,4,5-triphosphate binding; phosphatidylinositol-3,4-bisphosphate binding; phosphatidylinositol-4,5-bisphosphate binding; phosphoinositide binding; zinc ion binding

Biological Process: B cell differentiation; pre-B cell allelic exclusion; T cell differentiation in the thymus; V(D)J recombination

Disease: Combined Cellular And Humoral Immune Defects With Granulomas; Omenn Syndrome; Severe Combined Immunodeficiency, Autosomal Recessive, T Cell-negative, B Cell-negative, Nk Cell-positive

NCBI Summary:This gene encodes a protein that is involved in the initiation of V(D)J recombination during B and T cell development. This protein forms a complex with the product of the adjacent recombination activating gene 1, and this complex can form double-strand breaks by cleaving DNA at conserved recombination signal sequences. The recombination activating gene 1 component is thought to contain most of the catalytic activity, while the N-terminal of the recombination activating gene 2 component is thought to form a six-bladed propeller in the active core that serves as a binding scaffold for the tight association of the complex with DNA. A C-terminal plant homeodomain finger-like motif in this protein is necessary for interactions with chromatin components, specifically with histone H3 that is trimethylated at lysine 4. Mutations in this gene cause Omenn syndrome, a form of severe combined immunodeficiency associated with autoimmune-like symptoms. [provided by RefSeq, Jul 2008]
UniProt Code:P55895
NCBI GenInfo Identifier:2498830
NCBI Gene ID:5897
NCBI Accession:P55895.1
UniProt Secondary Accession:P55895,Q8TBL4, A8K9E9,
UniProt Related Accession:P55895
Molecular Weight:59,241 Da
NCBI Full Name:V(D)J recombination-activating protein 2
NCBI Synonym Full Names:recombination activating gene 2
NCBI Official Symbol:RAG2
NCBI Official Synonym Symbols:RAG-2
NCBI Protein Information:V(D)J recombination-activating protein 2
UniProt Protein Name:V(D)J recombination-activating protein 2
Protein Family:Seed allergenic protein
UniProt Gene Name:RAG2
UniProt Entry Name:RAG2_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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