Human MNAT1 ELISA Kit

Product Name:	Human MNAT1 ELISA Kit
Product Code:	HUFI01298
Size:	96 Assays
Alias:	MNAT1, CDK-activating kinase assembly factor MAT1, p35, RING finger protein MAT1, RING finger protein 66, Menage a trois, p36, CDK7, cyclin-H assembly factor, Cyclin-G1-interacting protein, CAP35, MAT1, RNF66, TFB3, CAP35, RNF66
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human MNAT1 concentrations in serum plasma and other biological fluids.
Sensitivity:	46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human MNAT1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human MNAT1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-104	96
EDTA plasma(n=5)	87-102	98
UFH plasma(n=5)	94-101	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human MNAT1 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)	89-97%	91-102%	92-105%
EDTA plasma(n=5)	86-99%	86-97%	83-100%
UFH plasma(n=5)	80-95%	80-99%	80-99%

CV(%):

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

P51948

UniProt Protein Function:	MNAT1: Stabilizes the cyclin H-CDK7 complex to form a functional CDK-activating kinase (CAK) enzymatic complex. CAK activates the cyclin-associated kinases CDK1, CDK2, CDK4 and CDK6 by threonine phosphorylation. CAK complexed to the core-TFIIH basal transcription factor activates RNA polymerase II by serine phosphorylation of the repetitive C-terminus domain (CTD) of its large subunit (POLR2A), allowing its escape from the promoter and elongation of the transcripts. Involved in cell cycle control and in RNA transcription by RNA polymerase II.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; Cell cycle regulation; Ubiquitin conjugating system; Ubiquitin ligase Chromosomal Location of Human Ortholog: 14q23 Cellular Component: nucleoplasm; holo TFIIH complex; cytoplasm Molecular Function:RNA polymerase subunit kinase activity; DNA-dependent ATPase activity; protein binding; zinc ion binding; protein N-terminus binding Biological Process: transcription from RNA polymerase II promoter; viral reproduction; positive regulation of viral transcription; positive regulation of smooth muscle cell proliferation; protein amino acid phosphorylation; regulation of gene expression, epigenetic; mRNA capping; negative regulation of gene expression, epigenetic; transcription-coupled nucleotide-excision repair; nucleotide-excision repair, DNA damage removal; protein complex assembly; G2/M transition of mitotic cell cycle; adult heart development; transcription initiation from RNA polymerase II promoter; ventricular system development; transcription from RNA polymerase I promoter; RNA elongation from RNA polymerase I promoter; termination of RNA polymerase I transcription; DNA repair; regulation of transcription from RNA polymerase II promoter; cell proliferation; nucleotide-excision repair; RNA elongation from RNA polymerase II promoter; gene expression; positive regulation of transcription from RNA polymerase II promoter; mitotic cell cycle; regulation of cyclin-dependent protein kinase activity; transcription initiation from RNA polymerase I promoter; response to calcium ion; negative regulation of apoptosis; G1/S transition of mitotic cell cycle
NCBI Summary:	The protein encoded by this gene, along with cyclin H and CDK7, forms the CDK-activating kinase (CAK) enzymatic complex. This complex activates several cyclin-associated kinases and can also associate with TFIIH to activate transcription by RNA polymerase II. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011]
UniProt Code:	P51948
NCBI GenInfo Identifier:	1708932
NCBI Gene ID:	4331
NCBI Accession:	P51948.1
UniProt Secondary Accession:	P51948,Q15817, Q6ICQ7, G3V1U8,
UniProt Related Accession:	P51948
Molecular Weight:	309
NCBI Full Name:	CDK-activating kinase assembly factor MAT1
NCBI Synonym Full Names:	MNAT CDK-activating kinase assembly factor 1
NCBI Official Symbol:	MNAT1
NCBI Official Synonym Symbols:	MAT1; TFB3; CAP35; RNF66
NCBI Protein Information:	CDK-activating kinase assembly factor MAT1; RING finger protein 66; RING finger protein MAT1; CDK7/cyclin-H assembly factor; cyclin G1 interacting protein; cyclin-G1-interacting protein; menage a trois 1 (CAK assembly factor); menage a trois homolog 1, cyclin H assembly factor
UniProt Protein Name:	CDK-activating kinase assembly factor MAT1
UniProt Synonym Protein Names:	CDK7/cyclin-H assembly factor; Cyclin-G1-interacting protein; Menage a trois; RING finger protein 66; RING finger protein MAT1; p35; p36
Protein Family:	CDK-activating kinase assembly factor
UniProt Gene Name:	MNAT1
UniProt Entry Name:	MAT1_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
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 Âµl 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 Âµl 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.