Rat Growth Factor Receptor Bound Protein 14 / GRB14 ELISA Kit

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SKU:
RTFI00833
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O88900
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
GRB14
Reactivity:
Rat
Frequently bought together:

Description

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Product Name:Rat GRB14 (Growth Factor Receptor Bound Protein 14) ELISA Kit
Product Code:RTFI00833
Size:96 Assays
Target:Rat GRB14
Alias:GRB14
Reactivity:Rat
Detection Method:Sandwich ELISA, Double Antibody
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Rat GRB14 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat GRB14 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)88-10294
EDTA plasma(n=5)85-9892
UFH plasma(n=5)87-10094
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat GRB14 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)88-101%88-103%90-97%
EDTA plasma(n=5)85-101%83-90%83-101%
UFH plasma(n=5)82-99%86-99%85-92%
Intra-Assay:CV <8%
Inter-Assay:CV <10%
Uniprot:O88900
UniProt Protein Function:GRB14: Adapter protein which modulates coupling of cell surface receptor kinases with specific signaling pathways. Binds to, and suppresses signals from, the activated insulin receptor (INSR). Potent inhibitor of insulin-stimulated MAPK3 phosphorylation. Plays a critical role regulating PDPK1 membrane translocation in response to insulin stimulation and serves as an adapter protein to recruit PDPK1 to activated insulin receptor, thus promoting PKB/AKT1 phosphorylation and transduction of the insulin signal. Belongs to the GRB7/10/14 family.
UniProt Protein Details:

Protein type:Adaptor/scaffold

Chromosomal Location of Human Ortholog: 3q21

Cellular Component: cytoplasm; endosome; Golgi apparatus; intracellular membrane-bound organelle; plasma membrane

Molecular Function:phosphoprotein binding; protein binding; SH3/SH2 adaptor activity

Biological Process: insulin receptor signaling pathway; negative regulation of insulin receptor signaling pathway

NCBI Summary:member of the Grb7 subfamily of adapters; functions as inhibitor in insulin-mediated pathways [RGD, Feb 2006]
UniProt Code:O88900
NCBI GenInfo Identifier:13928858
NCBI Gene ID:58844
NCBI Accession:NP_113811.1
UniProt Related Accession:O88900
Molecular Weight:34.6kDa
NCBI Full Name:growth factor receptor-bound protein 14
NCBI Synonym Full Names:growth factor receptor bound protein 14
NCBI Official Symbol:Grb14  
NCBI Protein Information:growth factor receptor-bound protein 14
UniProt Protein Name:Growth factor receptor-bound protein 14
UniProt Synonym Protein Names:GRB14 adapter protein
Protein Family:Growth factor receptor-bound protein
UniProt Gene Name:Grb14  
StepProcedure
1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4. Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37°C for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12. Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol
Serum:

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum and assay promptly or aliquot and store the samples at-80°C. Avoid multiple freeze-thaw cycles.

Plasma: Collect plasma using EDTA or heparin as an anti-coagulant. Centrifuge samples at 4°C for 15 mins at 1000 — g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid: Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell Culture Supernatant: Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell Lysates: Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenizein 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or-80°C.
Tissue Lysates: Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk: Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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