Human Snf1lk ELISA Kit (HUFI01318)

Product Name:	Human Snf1lk ELISA Kit
Product Code:	HUFI01318
Size:	96 Assays
Alias:	SIK1, Serine, threonine-protein kinase SIK1, SIK, SIK-1, SNF1LK, Salt-inducible protein kinase 1, Serine, threonine-protein kinase SNF1-like kinase 1, Serine, threonine-protein kinase SNF1LK
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human SIK1 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 SIK1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human SIK1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-105	96
EDTA plasma(n=5)	90-100	96
UFH plasma(n=5)	92-105	99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human SIK1 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)	90-103%	98-105%	86-92%
EDTA plasma(n=5)	89-100%	88-100%	82-101%
UFH plasma(n=5)	80-94%	87-98%	80-95%

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

P57059

UniProt Protein Function:	SIK: Serine/threonine-protein kinase involved in various processes such as cell cycle regulation, gluconeogenesis and lipogenesis regulation, muscle growth and differentiation and tumor suppression. Phosphorylates HDAC4, HDAC5, PPME1, SREBF1, TORC1/CRTC1 and TORC2/CRTC2. Acts as a tumor suppressor and plays a key role in p53/TP53-dependent anoikis, a type of apoptosis triggered by cell detachment: required for phosphorylation of p53/TP53 in response to loss of adhesion and is able to suppress metastasis. Part of a sodium-sensing signaling network, probably by mediating phosphorylation of PPME1: following increases in intracellular sodium, SIK1 is activated by CaMK1 and phosphorylates PPME1 subunit of protein phosphatase 2A (PP2A), leading to dephosphorylation of sodium/potassium-transporting ATPase ATP1A1 and subsequent increase activity of ATP1A1. Acts as a regulator of muscle cells by phosphorylating and inhibiting class II histone deacetylases HDAC4 and HDAC5, leading to promote expression of MEF2 target genes in myocytes. Also required during cardiomyogenesis by regulating the exit of cardiomyoblasts from the cell cycle via down-regulation of CDKN1C/p57Kip2. Acts as a regulator of hepatic gluconeogenesis by phosphorylating and repressing the CREB-specific coactivators TORC1/CRTC1 and TORC2/CRTC2, leading to inhibit CREB activity. Also regulates hepatic lipogenesis by phosphorylating and inhibiting SREBF1. Interacts with ATP1A1. Interacts (when phosphorylated on Thr-182 and Ser-186) with YWHAZ. Activated by phosphorylation on Thr-182. Also activated by phosphorylation on Thr-322 in response to increases in intracellular sodium in parallel with elevations in intracellular calcium through the reversible sodium/calcium exchanger. Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. AMPK subfamily.
UniProt Protein Details:	Protein type:EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Tumor suppressor; Kinase, protein; Protein kinase, CAMK; Motility/polarity/chemotaxis; CAMK group; CAMKL family; QIK subfamily Chromosomal Location of Human Ortholog: 21q22.3 Cellular Component: cytoplasm; nucleus Molecular Function:ATP binding; cAMP response element binding protein binding; magnesium ion binding; protein binding; protein kinase binding; protein serine/threonine kinase activity Biological Process: cardiac muscle cell differentiation; cellular response to hormone stimulus; entrainment of circadian clock by photoperiod; inhibition of CREB transcription factor; negative regulation of gluconeogenesis; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of cell differentiation; regulation of mitotic cell cycle; regulation of sodium ion transport Disease: Epileptic Encephalopathy, Early Infantile, 30
UniProt Code:	P57059
NCBI GenInfo Identifier:	59803093
NCBI Gene ID:	102724428
NCBI Accession:	P57059.2
UniProt Secondary Accession:	P57059,Q5R2V5, Q6ZNL8, Q86YJ2, A6NC84,
Molecular Weight:	84,902 Da
NCBI Full Name:	Serine/threonine-protein kinase SIK1
NCBI Official Symbol:	LOC102724428
NCBI Protein Information:	serine/threonine-protein kinase SIK1
UniProt Protein Name:	Serine/threonine-protein kinase SIK1
UniProt Synonym Protein Names:	Salt-inducible kinase 1; SIK-1; Serine/threonine-protein kinase SNF1-like kinase 1; Serine/threonine-protein kinase SNF1LK
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	SIK1
UniProt Entry Name:	SIK1_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.