Human STAT1 ELISA Kit (HUEB1912)- High Sensitivity

Product Name:	Human Signal transducer and activator of transcription 1-alpha/beta (STAT1) ELISA Kit
SKU:	HUEB1912
Size:	96T
Target:	Human Signal transducer and activator of transcription 1-alpha/beta (STAT1)
Synonyms:	Transcription factor ISGF-3 components p91/p84
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	31.2-2000pg/mL
Sensitivity:	15.61pg/mL

Intra CV:	Provided with the Kit
Inter CV:	Provided with the Kit
Linearity:	Provided with the Kit
Recovery:	Provided with the Kit
Function:	Signal transducer and transcription activator that mediates cellular responses to interferons (IFNs), cytokine KITLG/SCF and other cytokines and other growth factors. Following type I IFN (IFN-alpha and IFN-beta) binding to cell surface receptors, signaling via protein kinases leads to activation of Jak kinases (TYK2 and JAK1) and to tyrosine phosphorylation of STAT1 and STAT2. The phosphorylated STATs dimerize and associate with ISGF3G/IRF-9 to form a complex termed ISGF3 transcription factor, that enters the nucleus. ISGF3 binds to the IFN stimulated response element (ISRE) to activate the transcription of IFN-stimulated genes (ISG), which drive the cell in an antiviral state. In response to type II IFN (IFN-gamma), STAT1 is tyrosine- and serine-phosphorylated. It then forms a homodimer termed IFN-gamma-activated factor (GAF), migrates into the nucleus and binds to the IFN gamma activated sequence (GAS) to drive the expression of the target genes, inducing a cellular antiviral state. Becomes activated in response to KITLG/SCF and KIT signaling. May mediate cellular responses to activated FGFR1, FGFR2, FGFR3 and FGFR4.

Uniprot:	P42224
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Signal transducer and activator of transcription 1-alpha/beta
Sub Unit:	Isoform alpha homodimerizes upon IFN-gamma induced phosphorylation. Heterodimer with STAT2 upon IFN-alpha/beta induced phosphorylation. The heterodimer STAT1:STAT2 forms the interferon-stimulated gene factor 3 complex (ISGF3) with IRF9. Interacts (phosphorylated at Ser 727) with PIAS1 (dimethylated on arginine); the interaction results in release of STAT1 from its target gene. Interacts with IFNAR1; the interaction requires the phosphorylation of IFNAR1 at 'Tyr-466'. Interacts with IFNAR2, NMI, PTK2/FAK1 and SRC. Interacts with ERBB4 (phosphorylated). Interacts with Sendai virus C', C, Y1 and Y2 proteins, Nipah virus P, V and W proteins, and rabies virus phosphoprotein preventing activation of ISRE and GAS promoter. Interacts with HCV core protein; the interaction results in STAT1 degradation.
Research Area:	Cancer
Subcellular Location:	Cytoplasm Nucleus Translocated into the nucleus upon tyrosine phosphorylation and dimerization, in response to IFN-gamma and signaling by activated FGFR1, FGFR2, FGFR3 or FGFR4.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	STAT1: transcription factor of the STAT family. Phosphorylated and activated by receptor-associated kinases downstream of certain receptor tyrosine kinases, GPCRs, and receptors for various interleukins and interferons. Forms homo- or heterodimers that translocate into the nucleus where they regulate transcription. Two alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 2q32.2 Cellular Component: axon; cytoplasm; cytosol; dendrite; nuclear chromatin; nucleolus; nucleoplasm; nucleus; perinuclear region of cytoplasm Molecular Function:double-stranded DNA binding; enzyme binding; identical protein binding; protein binding; protein homodimerization activity; signal transducer activity; transcription factor activity; tumor necrosis factor receptor binding Biological Process: apoptosis; blood circulation; defense response to virus; endothelial cell migration; JAK-STAT cascade; negative regulation of angiogenesis; negative regulation of endothelial cell proliferation; negative regulation of I-kappaB kinase/NF-kappaB cascade; negative regulation of transcription from RNA polymerase II promoter; negative regulation of viral protein levels in host cell; positive regulation of mesenchymal cell proliferation; positive regulation of smooth muscle cell proliferation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of apoptosis; regulation of transcription from RNA polymerase II promoter; response to cAMP; response to cytokine stimulus; response to peptide hormone stimulus; transcription, DNA-dependent; tumor necrosis factor-mediated signaling pathway; viral reproduction Disease: Immunodeficiency 31a; Immunodeficiency 31b; Immunodeficiency 31c
NCBI Summary:	The protein encoded by this gene is a member of the STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein can be activated by various ligands including interferon-alpha, interferon-gamma, EGF, PDGF and IL6. This protein mediates the expression of a variety of genes, which is thought to be important for cell viability in response to different cell stimuli and pathogens. Two alternatively spliced transcript variants encoding distinct isoforms have been described. [provided by RefSeq, Jul 2008]
UniProt Code:	P42224
NCBI GenInfo Identifier:	2507413
NCBI Gene ID:	6772
NCBI Accession:	P42224.2
UniProt Secondary Accession:	P42224,Q53S88, Q53XW4, Q68D00, Q9UDL5, A8K989, B2RCA0 D2KFR8, D3DPI7,
UniProt Related Accession:	P42224
Molecular Weight:
NCBI Full Name:	Signal transducer and activator of transcription 1-alpha/beta
NCBI Synonym Full Names:	signal transducer and activator of transcription 1
NCBI Official Symbol:	STAT1
NCBI Official Synonym Symbols:	CANDF7; IMD31A; IMD31B; IMD31C; ISGF-3; STAT91
NCBI Protein Information:	signal transducer and activator of transcription 1-alpha/beta
UniProt Protein Name:	Signal transducer and activator of transcription 1-alpha/beta
UniProt Synonym Protein Names:	Transcription factor ISGF-3 components p91/p84
Protein Family:	Signal transducer and activator of transcription
UniProt Gene Name:	STAT1
UniProt Entry Name:	STAT1_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA	Antibodies
Human STAT1 ELISA Kit	Anti-STAT1 Antibody (CAB0027)
STAT1 (Phospho-Ser727) Transcription Factor Activity Assay	Anti-STAT1 Mouse Monoclonal Antibody (CAB10100)
STAT1 (Phospho-Tyr701) Transcription Factor Activity Assay	Anti-STAT1 Antibody (CAB12075)
STAT1 Transcription Factor Activity Assay	Anti-STAT1 Antibody (CAB12330)
	Anti-STAT1 Antibody (CAB19563)