Mouse PKB/Protein Kinase B ELISA Kit (MOFI01053)

Product Name:	Mouse PKB/Protein Kinase B ELISA Kit
Product Code:	MOFI01053
Size:	96 Assays
Alias:	PKB, PKB alpha, PRKBA, RAC-alpha, Protein kinase B, 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
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse PKB concentrations in serum plasma and other biological fluids.
Sensitivity:	0.469ng/ml
Range:	0.781-50ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse PKB and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse PKB in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-102	95
EDTA plasma(n=5)	92-97	94
UFH plasma(n=5)	92-103	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse 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)	85-98%	94-103%	87-100%
EDTA plasma(n=5)	83-98%	86-98%	83-101%
UFH plasma(n=5)	80-98%	81-97%	85-100%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P31750

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; Oncoprotein; Kinase, protein; Protein kinase, AGC; Protein kinase, Ser/Thr (non-receptor); AGC group; AKT family Cellular Component: cytoplasm; cytosol; intercellular junction; microtubule cytoskeleton; mitochondrion; nucleus; plasma membrane; protein complex; spindle; vesicle Molecular Function:ATP binding; enzyme binding; GTPase activating protein binding; identical protein binding; kinase activity; nitric-oxide synthase regulator activity; phosphatidylinositol-3,4,5-triphosphate binding; phosphatidylinositol-3,4-bisphosphate binding; protein binding; protein kinase activity; protein kinase binding; protein kinase C binding; protein phosphatase 2A binding; protein serine/threonine kinase activity; protein serine/threonine/tyrosine kinase activity Biological Process: activated T cell apoptosis; aging; anagen; apoptotic mitochondrial changes; cell projection organization and biogenesis; cellular response to insulin stimulus; cytoskeleton organization and biogenesis; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; germ cell development; glucose homeostasis; glucose metabolic process; glucose transport; glycogen biosynthetic process; glycogen metabolic process; inflammatory response; insulin receptor signaling pathway; insulin-like growth factor receptor signaling pathway; lipopolysaccharide-mediated signaling pathway; maternal placenta development; myelin maintenance in the peripheral nervous system; negative regulation of apoptosis; negative regulation of autophagy; negative regulation of caspase activity; negative regulation of cell size; negative regulation of fatty acid beta-oxidation; negative regulation of JNK cascade; negative regulation of protein kinase activity; negative regulation of proteolysis; osteoblast differentiation; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; phosphorylation; positive regulation of apoptosis; positive regulation of blood vessel endothelial cell migration; positive regulation of cell growth; positive regulation of cellular protein metabolic process; positive regulation of endodeoxyribonuclease activity; positive regulation of endothelial cell proliferation; positive regulation of fat cell differentiation; positive regulation of glucose import; positive regulation of glycogen biosynthetic process; positive regulation of lipid biosynthetic process; positive regulation of nitric oxide biosynthetic process; positive regulation of nitric-oxide synthase activity; positive regulation of peptidyl-serine phosphorylation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; positive regulation of protein amino acid phosphorylation; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of vasoconstriction; protein amino acid phosphorylation; protein catabolic process; protein import into nucleus, translocation; protein kinase B signaling cascade; protein ubiquitination; regulation of cell migration; regulation of glycogen biosynthetic process; regulation of myelination; regulation of protein localization; response to DNA damage stimulus; response to food; response to hormone stimulus; response to organic substance; signal transduction; spinal cord development; striated muscle cell differentiation; translation
NCBI Summary:	This gene encodes the founding member of the Akt serine-threonine protein kinase gene family that also includes Akt2 and Akt3. This kinase is a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway that mediates the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). It is activated through recruitment to cellular membranes by PI3K lipid products and by phosphorylation by 3-phosphoinositide dependent kinase-1. It then further phosphorylates different downstream proteins in response to various extracellular signals and thus plays a pivotal role in mediating a variety of cellular processes, such as glucose metabolism, glycogen biosynthesis, protein synthesis and turn over, inflammatory response, cell survival (anti-apoptosis) and development. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2009]
UniProt Code:	P31750
NCBI GenInfo Identifier:	341940204
NCBI Gene ID:	11651
NCBI Accession:	P31750.2
UniProt Secondary Accession:	P31750,Q62274, Q6GSA6,
UniProt Related Accession:	P31750
Molecular Weight:	55,707 Da
NCBI Full Name:	RAC-alpha serine/threonine-protein kinase
NCBI Synonym Full Names:	thymoma viral proto-oncogene 1
NCBI Official Symbol:	Akt1
NCBI Official Synonym Symbols:	Akt; PKB; Rac; PKB/Akt; PKBalpha
NCBI Protein Information:	RAC-alpha serine/threonine-protein kinase
UniProt Protein Name:	RAC-alpha serine/threonine-protein kinase
UniProt Synonym Protein Names:	AKT1 kinase; Protein kinase B; PKB; Protein kinase B alpha; PKB alpha; Proto-oncogene c-Akt; RAC-PK-alpha; Thymoma viral proto-oncogene
Protein Family:	Potassium channel
UniProt Gene Name:	Akt1
UniProt Entry Name:	AKT1_MOUSE

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

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 clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum 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 anticoagulant. 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 & homogenize in 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.