Human eIF3a (Eukaryotic Translation Initiation Factor 3a) ELISA Kit (HUES03166)

SKU:
HUES03166
€539

Description

ELISA Kit Technical ManualMSDS

Human eIF3a (Eukaryotic Translation Initiation Factor 3a) 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 eIF3a . 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 eIF3a 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 eIF3a, 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 eIF3a. The concentration of Human eIF3a 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 Range62.50-4000 pg/mL
Sensitivity37.50 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human eIF3a in samples. No significant cross-reactivity or interference between Human eIF3a 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
40002.411
2.431
2.4212.344
20001.576
1.612
1.5941.517
10000.896
0.864
0.880.803
5000.503
0.519
0.5110.434
2500.269
0.247
0.2580.181
1250.175
0.173
0.1740.097
62.500.126
0.128
0.1270.05
00.077
0.077
0.077--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human eIF3a were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human eIF3a were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)184.74465.521591.59171.35455.871646.55
Standard deviation12.5620.0263.359.1724.4375.25
C V (%)6.804.303.985.355.364.57

Recovery

The recovery of Human eIF3a 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)89-10294
EDTA plasma (n=5)92-10698
Cell culture media (n=5)89-10196

Linearity

Samples were spiked with high concentrations of Human eIF3a 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 (%)95-10783-9599-115
Average (%)10090105
1:4Range (%)88-10283-9786-97
Average (%)938992
1:8Range (%)93-10582-9684-96
Average (%)998789
1:16Range (%)92-10785-9784-97
Average (%)1009191

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 eIF3a (Eukaryotic Translation Initiation Factor 3a) ELISA Kit (HUES03166) 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 eIF3a (Eukaryotic Translation Initiation Factor 3a) ELISA Kit (HUES03166) Protein Information

UniProt Protein Function:eIF3-theta: Component of the eukaryotic translation initiation factor 3 (eIF-3) complex, which is required for several steps in the initiation of protein synthesis. The eIF-3 complex associates with the 40S ribosome and facilitates the recruitment of eIF-1, eIF-1A, eIF-2:GTP:methionyl-tRNAi and eIF-5 to form the 43S preinitiation complex (43S PIC). The eIF-3 complex stimulates mRNA recruitment to the 43S PIC and scanning of the mRNA for AUG recognition. The eIF-3 complex is also required for disassembly and recycling of posttermination ribosomal complexes and subsequently prevents premature joining of the 40S and 60S ribosomal subunits prior to initiation. Interacts with EIF4G1. Component of the eukaryotic translation initiation factor 3 (eIF-3) complex, which is composed of 13 subunits: EIF3A, EIF3B, EIF3C, EIF3D, EIF3E, EIF3F, EIF3G, EIF3H, EIF3I, EIF3J, EIF3K, EIF3L and EIF3M. The eIF-3 complex appears to include 3 stable modules: module A is composed of EIF3A, EIF3B, EIF3G and EIF3I; module B is composed of EIF3F, EIF3H, and EIF3M; and module C is composed of EIF3C, EIF3D, EIF3E, EIF3L and EIF3K. EIF3C of module C binds EIF3B of module A and EIF3H of module B, thereby linking the three modules. EIF3J is a labile subunit that binds to the eIF-3 complex via EIF3B. The eIF-3 complex interacts with RPS6KB1 under conditions of nutrient depletion. Mitogenic stimulation leads to binding and activation of a complex composed of MTOR and RPTOR, leading to phosphorylation and release of RPS6KB1 and binding of EIF4B to eIF-3. Also interacts with KRT7 and PIWIL2. Belongs to the eIF-3 subunit A family.
UniProt Protein Details:

Protein type:Translation; RNA-binding; Translation initiation

Chromosomal Location of Human Ortholog: 10q26

Cellular Component: cytoplasm; cytosol; eukaryotic translation initiation factor 3 complex; membrane; nucleolus; nucleus

Molecular Function:protein binding; translation initiation factor activity

Biological Process: formation of translation initiation complex; translational initiation

UniProt Code:Q14152
NCBI GenInfo Identifier:6685537
NCBI Gene ID:8661
NCBI Accession:Q14152.1
UniProt Secondary Accession:Q14152,O00653, Q15778, B1AMV5, B4DYS1, F5H335,
UniProt Related Accession:Q14152
Molecular Weight:162,636 Da
NCBI Full Name:Eukaryotic translation initiation factor 3 subunit A
NCBI Synonym Full Names:eukaryotic translation initiation factor 3 subunit A
NCBI Official Symbol:EIF3A
NCBI Official Synonym Symbols:EIF3; P167; p180; p185; TIF32; EIF3S10; eIF3-p170; eIF3-theta
NCBI Protein Information:eukaryotic translation initiation factor 3 subunit A
UniProt Protein Name:Eukaryotic translation initiation factor 3 subunit A
UniProt Synonym Protein Names:Eukaryotic translation initiation factor 3 subunit 10
Protein Family:Eukaryotic translation initiation factor
UniProt Gene Name:EIF3A
UniProt Entry Name:EIF3A_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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