Rat Protein Kinase B/PKB/AKT ELISA Kit (RTFI01064)

Product Name:	Rat Protein Kinase B/PKB/AKT ELISA Kit
Product Code:	RTFI01064
Size:	96 Assays
Target:	Rat PKB
Alias:	Akt1, PKB alpha, PRKBA, RAC-alpha, AKT, PRKBA, Protein kinase B, PKB, Proto-oncogene c-Akt, rac protein kinase alpha, RAC-ALPHA, RAC-alpha serine, threonine-protein kinase, RAC-PK-alpha, RACPKB-ALPHA, v-akt murine thymoma viral oncogene homolog 1
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 PKB and the recovery rates were calculated by comparing the measured value to the expected amount of Rat PKB in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-102	94
EDTA plasma(n=5)	91-103	99
UFH plasma(n=5)	88-104	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat PKB and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	87-105%	88-103%	88-104%
EDTA plasma(n=5)	83-99%	82-100%	84-97%
UFH plasma(n=5)	81-100%	80-91%	87-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P47196

UniProt Protein Function:	Akt1: an oncogenic AGC kinase that plays a critical role in regulating cell survival and metabolism in many different signaling pathways. Dual phosphorylation is required for its activation. T308 is phosphorylated by PDK1 in the PI3 kinase pathway, and S473 is phosphorylated by mTOR in the mTORC2 pathway. The 'Lys-63'-linked ubiquitination of AKT1 by TRAF6 is important for its translocation to the plasma membrane, phosphorylation, and activation. When Akt is fully phosphorylated it translocates into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its proteosomal degradation. Hyperactive or overexpressed in a number of cancers including breast, prostate, lung, pancreatic, liver, ovarian and colorectal. Over 160 protein substrates are known including many that regulate transcription, metabolism, apoptosis, cell cycle, and growth.
UniProt Protein Details:	Protein type:EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; Oncoprotein; Kinase, protein; AGC group; AKT family Chromosomal Location of Human Ortholog: 14q32.32 Cellular Component: nucleoplasm; microtubule cytoskeleton; mitochondrion; cytoplasm; plasma membrane; spindle; intercellular junction; nucleus; cytosol Molecular Function:identical protein binding; protein serine/threonine kinase activity; protein binding; phosphatidylinositol-3,4,5-triphosphate binding; enzyme binding; protein kinase C binding; nitric-oxide synthase regulator activity; protein serine/threonine/tyrosine kinase activity; kinase activity; phosphatidylinositol-3,4-bisphosphate binding; ATP binding; protein kinase activity Biological Process: negative regulation of JNK cascade; positive regulation of nitric oxide biosynthetic process; regulation of myelination; nerve growth factor receptor signaling pathway; protein ubiquitination; glucose homeostasis; regulation of cell migration; protein amino acid phosphorylation; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; germ cell development; positive regulation of glucose import; cell projection organization and biogenesis; protein catabolic process; maternal placenta development; response to food; platelet activation; glycogen biosynthetic process; fibroblast growth factor receptor signaling pathway; positive regulation of nitric-oxide synthase activity; positive regulation of blood vessel endothelial cell migration; glucose metabolic process; positive regulation of lipid biosynthetic process; positive regulation of cell growth; insulin-like growth factor receptor signaling pathway; cellular response to insulin stimulus; response to heat; T cell costimulation; positive regulation of fat cell differentiation; negative regulation of protein kinase activity; striated muscle cell differentiation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of endothelial cell proliferation; positive regulation of transcription factor activity; response to oxidative stress; regulation of nitric-oxide synthase activity; negative regulation of apoptosis; negative regulation of autophagy; negative regulation of fatty acid beta-oxidation; translation; apoptosis; protein amino acid autophosphorylation; regulation of glycogen biosynthetic process; positive regulation of cellular protein metabolic process; positive regulation of glycogen biosynthetic process; negative regulation of cell size; negative regulation of caspase activity; glucose transport; signal transduction; nitric oxide metabolic process; regulation of translation; apoptotic mitochondrial changes; protein kinase B signaling cascade; inflammatory response; nitric oxide biosynthetic process; cell differentiation; activated T cell apoptosis; aging; negative regulation of proteolysis; epidermal growth factor receptor signaling pathway; phosphoinositide-mediated signaling; myelin maintenance in the peripheral nervous system; protein modification process; endocrine pancreas development; positive regulation of peptidyl-serine phosphorylation; osteoblast differentiation; cell proliferation; G-protein coupled receptor protein signaling pathway; peptidyl-serine phosphorylation; protein import into nucleus, translocation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; insulin receptor signaling pathway; positive regulation of vasoconstriction; innate immune response; gene expression; positive regulation of protein amino acid phosphorylation; blood coagulation; vascular endothelial growth factor receptor signaling pathway; phosphorylation; hyaluronan metabolic process Disease: Schizophrenia; Cowden Syndrome 6; Proteus Syndrome; Breast Cancer; Ovarian Cancer
NCBI Summary:	The serine-threonine protein kinase encoded by the AKT1 gene is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1. It was shown that the activation occurs through phosphatidylinositol 3-kinase. In the developing nervous system AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates components of the apoptotic machinery. Mutations in this gene have been associated with the Proteus syndrome. Multiple alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Jul 2011]
UniProt Code:	P47196
NCBI GenInfo Identifier:	62241013
NCBI Gene ID:	207
NCBI Accession:	NP_001014431.1
UniProt Secondary Accession:	P47196,P31751, Q9Y243, P31750, Q60823, Q9WUA6, P47196 P47197, Q63484,
UniProt Related Accession:	P31749
Molecular Weight:	56kDa
NCBI Full Name:	RAC-alpha serine/threonine-protein kinase
NCBI Synonym Full Names:	AKT serine/threonine kinase 1
NCBI Official Symbol:	AKT1
NCBI Official Synonym Symbols:	AKT; PKB; RAC; CWS6; PRKBA; PKB-ALPHA; RAC-ALPHA
NCBI Protein Information:	RAC-alpha serine/threonine-protein kinase
UniProt Protein Name:	RAC-alpha serine/threonine-protein kinase
UniProt Synonym Protein Names:	Protein kinase B; PKB; Protein kinase B alpha; PKB alpha; Proto-oncogene c-Akt; RAC-PK-alpha
Protein Family:	AKT-interacting protein
UniProt Gene Name:	AKT1
UniProt Entry Name:	AKT1_HUMAN

Step	Procedure
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.