Human LATS1 ELISA Kit (HUFI03322)- High Sensitivity

Product Name:	Human LATS1 (Serine/threonine-protein kinase LATS1) ELISA Kit
Product Code:	HUFI03322
Size:	96 Assays
Alias:	LATS1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human LATS1 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 Human LATS1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human LATS1 in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human LATS1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
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

O95835

UniProt Protein Function:	LATS1: an AGC kinase of the NDR family of kinases. Localized to the mitotic apparatus and specifically phosphorylated during mitosis. A likely tumor suppressor which plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G1 tetraploidy checkpoint. Negatively regulates G2/M transition by down-regulating CDC2 kinase activity, causing G2 arrest. Involved in the control of p53 expression. Affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1. Complexes with CDC2 in early mitosis. LATS1-associated CDC2 has no mitotic cyclin partner and no apparent kinase activity. Binds phosphorylated zyxin, locating this protein to the mitotic spindle and suggesting a role for actin regulatory proteins during mitosis. Binds to and colocalizes with LIMK1 at the actomyosin contractile ring during cytokinesis. Knockout mice are susceptible to soft-tissue sarcomas and sensitive to chemical carcinogenesis. Human soft tissue sarcomas have downregulated, mutated, and/or hypermethylated LATS1. Transgenic expression blocks anchorage independent growth in culture and tumor growth in xenografts. Two alternatively spliced isoforms of the human proteinhave been described.
UniProt Protein Details:	Protein type:Protein kinase, AGC; Tumor suppressor; EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; AGC group; NDR family Chromosomal Location of Human Ortholog: 6q25.1 Cellular Component: cytosol; microtubule organizing center; spindle pole Molecular Function:ATP binding; magnesium ion binding; protein binding; protein kinase binding; protein serine/threonine kinase activity Biological Process: cell division; cytoplasmic sequestering of protein; G1/S transition of mitotic cell cycle; G2/M transition of mitotic cell cycle; hormone-mediated signaling; inner cell mass cell fate commitment; inner cell mass cellular morphogenesis; keratinocyte differentiation; mitosis; negative regulation of cyclin-dependent protein kinase activity; peptidyl-serine phosphorylation; positive regulation of apoptosis; positive regulation of peptidyl-serine phosphorylation; protein amino acid phosphorylation; regulation of actin filament polymerization; regulation of organ growth; regulation of protein complex assembly; sister chromatid segregation
NCBI Summary:	The protein encoded by this gene is a putative serine/threonine kinase that localizes to the mitotic apparatus and complexes with cell cycle controller CDC2 kinase in early mitosis. The protein is phosphorylated in a cell-cycle dependent manner, with late prophase phosphorylation remaining through metaphase. The N-terminal region of the protein binds CDC2 to form a complex showing reduced H1 histone kinase activity, indicating a role as a negative regulator of CDC2/cyclin A. In addition, the C-terminal kinase domain binds to its own N-terminal region, suggesting potential negative regulation through interference with complex formation via intramolecular binding. Biochemical and genetic data suggest a role as a tumor suppressor. This is supported by studies in knockout mice showing development of soft-tissue sarcomas, ovarian stromal cell tumors and a high sensitivity to carcinogenic treatments. Two protein-coding transcripts and one non-protein coding transcript have been found for this gene. [provided by RefSeq, Jul 2012]
UniProt Code:	O95835
NCBI GenInfo Identifier:	52783106
NCBI Gene ID:	9113
NCBI Accession:	O95835.1
UniProt Secondary Accession:	O95835,Q6PKD0,
UniProt Related Accession:	O95835
Molecular Weight:
NCBI Full Name:	Serine/threonine-protein kinase LATS1
NCBI Synonym Full Names:	large tumor suppressor kinase 1
NCBI Official Symbol:	LATS1
NCBI Official Synonym Symbols:	wts; WARTS
NCBI Protein Information:	serine/threonine-protein kinase LATS1
UniProt Protein Name:	Serine/threonine-protein kinase LATS1
UniProt Synonym Protein Names:	Large tumor suppressor homolog 1; WARTS protein kinase; h-warts
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	LATS1
UniProt Entry Name:	LATS1_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 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.