Product Name:	Mouse c-Myc ELISA Kit
Product Code:	MOFI00729
Size:	96 Assays
Alias:	c-myc, Myc, Myc2, Niard, Nird, avian myelocytomatosis viral oncogene homolog, BHLHE39, bHLHe39MRTL, Class E basic helix-loop-helix protein 39, c-Myc, myc proto-oncogene protein, myc-related translation, localization regulatory factor, Proto-oncogene c-Myc, Transcription factor p64, v-myc avian myelocytomatosis viral oncogene homolog, v-myc myelocytomatosis viral oncogene homolog, avian
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse c-myc concentrations in serum plasma and other biological fluids.
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 Mouse c-myc and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse c-myc in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-103	91
EDTA plasma(n=5)	97-103	100
UFH plasma(n=5)	90-95	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse c-myc 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)	87-102%	89-96%	87-104%
EDTA plasma(n=5)	83-90%	84-101%	91-100%
UFH plasma(n=5)	80-100%	96-99%	80-99%

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

P01108

UniProt Protein Function:	Myc: a proto-oncogenic transcription factor that plays a role in cell proliferation, apoptosis and in the development of human tumors. Seems to activate the transcription of growth-related genes.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor; Nucleolus; Oncoprotein Cellular Component: axon; cytoplasm; mitochondrion; nuclear body; nucleolus; nucleoplasm; nucleus; perinuclear region of cytoplasm; protein complex; spindle Molecular Function:DNA binding; double-stranded DNA binding; protein binding; protein complex binding; protein dimerization activity; protein heterodimerization activity; sequence-specific DNA binding; transcription factor activity; transcription factor binding; ubiquitin protein ligase binding Biological Process: amino acid transport; B cell apoptosis; caspase activation; cell cycle arrest; cell proliferation; cellular iron ion homeostasis; chromatin remodeling; chromosome organization and biogenesis; detection of mechanical stimulus involved in sensory perception of sound; DNA damage response, signal transduction resulting in induction of apoptosis; glucose metabolic process; inner mitochondrial membrane organization and biogenesis; lactic acid secretion; MAPKKK cascade; middle ear morphogenesis; negative regulation of cell division; negative regulation of fibroblast proliferation; negative regulation of glucose import; negative regulation of monocyte differentiation; negative regulation of protein binding; negative regulation of transcription from RNA polymerase II promoter; pigmentation; positive regulation of B cell apoptosis; positive regulation of caspase activity; positive regulation of catalytic activity; positive regulation of cell cycle; positive regulation of cell proliferation; positive regulation of DNA binding; positive regulation of epithelial cell proliferation; positive regulation of fibroblast proliferation; positive regulation of glycolysis; positive regulation of mesenchymal cell proliferation; positive regulation of smooth muscle cell migration; positive regulation of smooth muscle cell proliferation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; protein processing; pyruvate transport; re-entry into mitotic cell cycle; regulation of apoptosis; regulation of gene expression; regulation of mitotic cell cycle; regulation of oxidative phosphorylation; regulation of telomere maintenance; regulation of transcription, DNA-dependent; response to alkaloid; response to DNA damage stimulus; response to gamma radiation; response to radiation; skeletal morphogenesis; transcription from RNA polymerase II promoter; transcription initiation; transcription, DNA-dependent; transformation of host cell by virus; ureteric bud branching; Wnt receptor signaling pathway; Wnt receptor signaling pathway through beta-catenin
NCBI Summary:	The protein encoded by this gene is a multifunctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation. It functions as a transcription factor that regulates transcription of specific target genes. Mutations, overexpression, rearrangement and translocation of this gene have been associated with a variety of hematopoietic tumors, leukemias and lymphomas, including Burkitt lymphoma, in human. There is evidence to show that alternative translation initiations from an upstream, in-frame non-AUG (CUG) and a downstream AUG start site result in the production of two isoforms with distinct N-termini, in human and mouse. Under conditions of stress, such as high cell densities and methionine deprivation, there is a specific and dramatic increase in the synthesis of the non-AUG initiated protein, suggesting its importance in times of adversity. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Apr 2010]
UniProt Code:	P01108
NCBI GenInfo Identifier:	293629264
NCBI Gene ID:	17869
NCBI Accession:	NP_001170823.1
UniProt Secondary Accession:	P01108,P70247, Q3UM70, Q61422,
UniProt Related Accession:	P01108
Molecular Weight:	76.0 kDa
NCBI Full Name:	myc proto-oncogene protein isoform b
NCBI Synonym Full Names:	myelocytomatosis oncogene
NCBI Official Symbol:	Myc
NCBI Official Synonym Symbols:	Myc2; Nird; Niard; bHLHe39; AU016757
NCBI Protein Information:	myc proto-oncogene protein
UniProt Protein Name:	Myc proto-oncogene protein
UniProt Synonym Protein Names:	Proto-oncogene c-Myc; Transcription factor p64
Protein Family:	Myc protein
UniProt Gene Name:	Myc
UniProt Entry Name:	MYC_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.