Human RPS6K beta1 (Ribosomal Protein S6 Kinase Beta 1) ELISA Kit (HUES02285)

SKU:
HUES02285
€539

Description

ELISA Kit Technical ManualMSDS

Human RPS6K beta1 (Ribosomal Protein S6 Kinase Beta 1) 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 RPS6K beta1 . 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 RPS6K beta1 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 RPS6K beta1, 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 RPS6K beta1. The concentration of Human RPS6K beta1 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 RPS6K beta1 in samples. No significant cross-reactivity or interference between Human RPS6K beta1 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
102.491
2.497
2.4942.439
51.542
1.59
1.5661.511
2.50.916
0.894
0.9050.85
1.250.464
0.488
0.4760.421
0.630.254
0.228
0.2410.186
0.320.163
0.155
0.1590.104
0.160.101
0.115
0.1080.053
00.047
0.063
0.055--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human RPS6K beta1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human RPS6K beta1 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.510.963.370.490.953.51
Standard deviation0.030.040.130.030.040.17
C V (%)5.884.173.866.124.214.84

Recovery

The recovery of Human RPS6K beta1 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)87-9892
EDTA plasma (n=5)88-9993
Cell culture media (n=5)96-110101

Linearity

Samples were spiked with high concentrations of Human RPS6K beta1 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 (%)97-11386-9991-105
Average (%)1059197
1:4Range (%)89-10286-9984-99
Average (%)959290
1:8Range (%)93-10981-9484-99
Average (%)998891
1:16Range (%)93-10683-9386-100
Average (%)998791

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 RPS6K beta1 (Ribosomal Protein S6 Kinase Beta 1) ELISA Kit (HUES02285) 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 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL 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 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.
    7. 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.
    8. Add 100 µL 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 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.
    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 RPS6K beta1 (Ribosomal Protein S6 Kinase Beta 1) ELISA Kit (HUES02285) Protein Information

UniProt Protein Function:p70S6K: an AGC kinase of the RSK family that is required for cell growth and G1 cell cycle progression. Is phosphorylated and activated by mTOR in mitogenic pathways downstream of phosphoinositide 3 kinase (PI3K). Phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs. Activity is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains. Mouse knockout shows symptoms of insulin resistance, and increased insulin senstivity, resulting in protection against diet-induced obesity. Protein expression and activation upregulated in colon adenocarcinoma cell lines. Increased expression in breast cancer correlated with poor survival. Selectively amplified and overexpressed within the 17q23 breast cancer amplicon. Two isoforms produced by alternative initiation have been reported.
UniProt Protein Details:

Protein type:EC 2.7.11.1; Translation; Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; Kinase, protein; AGC group; RSK family; p70 subfamily

Chromosomal Location of Human Ortholog: 17q23.1

NCBI Summary:This gene encodes a member of the ribosomal S6 kinase family of serine/threonine kinases. The encoded protein responds to mTOR (mammalian target of rapamycin) signaling to promote protein synthesis, cell growth, and cell proliferation. Activity of this gene has been associated with human cancer. Alternatively spliced transcript variants have been observed. The use of alternative translation start sites results in isoforms with longer or shorter N-termini which may differ in their subcellular localizations. There are two pseudogenes for this gene on chromosome 17. [provided by RefSeq, Jan 2013]
UniProt Code:P23443
NCBI GenInfo Identifier:54041234
NCBI Gene ID:6198
NCBI Accession:P23443.2
UniProt Secondary Accession:P23443,Q7Z721, B2R779, B4DLT4, B4DTG1, E7ESB8, F6UYM1
UniProt Related Accession:P23443
Molecular Weight:525
NCBI Full Name:Ribosomal protein S6 kinase beta-1
NCBI Synonym Full Names:ribosomal protein S6 kinase, 70kDa, polypeptide 1
NCBI Official Symbol:RPS6KB1
NCBI Official Synonym Symbols:S6K; PS6K; S6K1; STK14A; p70-S6K; p70 S6KA; p70-alpha; S6K-beta-1; p70(S6K)-alpha
NCBI Protein Information:ribosomal protein S6 kinase beta-1; p70 S6 kinase, alpha; ribosomal protein S6 kinase I; serine/threonine kinase 14 alpha; serine/threonine-protein kinase 14A
UniProt Protein Name:Ribosomal protein S6 kinase beta-1
UniProt Synonym Protein Names:70 kDa ribosomal protein S6 kinase 1; P70S6K1; p70-S6K 1; Ribosomal protein S6 kinase I; Serine/threonine-protein kinase 14A; p70 ribosomal S6 kinase alpha; p70 S6 kinase alpha; p70 S6K-alpha; p70 S6KA
Protein Family:Ribosomal protein S6 kinase
UniProt Gene Name:RPS6KB1
UniProt Entry Name:KS6B1_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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