Human STAT3 ELISA Kit (HUEB0089)- High Sensitivity

Product Name:	Human Signal transducer and activator of transcription 3 (STAT3) ELISA Kit
SKU:	HUEB0089
Size:	96T
Target:	Human Signal transducer and activator of transcription 3 (STAT3)
Synonyms:	Acute-phase response factor, APRF
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.12ng/mL

Intra CV:

5.2%

Inter CV:

9.4%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	107-117%	116-126%	105-116%	90-90%
EDTA Plasma(N=5)	89-102%	96-109%	109-119%	88-100%
Heparin Plasma(N=5)	99-109%	106-116%	109-119%	88-97%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	88	82-94
Plasma	90	84-96

Function:

Signal transducer and transcription activator that mediates cellular responses to interleukins, KITLG/SCF, LEP and other growth factors (PubMed:10688651, PubMed:12359225, PubMed:12873986, PubMed:15194700, PubMed:17344214, PubMed:18242580, PubMed:23084476). Once activated, recruits coactivators, such as NCOA1 or MED1, to the promoter region of the target gene (PubMed:17344214). May mediate cellular responses to activated FGFR1, FGFR2, FGFR3 and FGFR4 (PubMed:12873986). Binds to the interleukin-6 (IL-6)-responsive elements identified in the promoters of various acute-phase protein genesPubMed:12359225. Activated by IL31 through IL31RA (PubMed:15194700). Acts as a regulator of inflammatory response by regulating differentiation of naive CD4(+) T-cells into T-helper Th17 or regulatory T-cells (Treg): deacetylation and oxidation of lysine residues by LOXL3, leads to disrupt STAT3 dimerization and inhibit its transcription activity (PubMed:28065600). Involved in cell cycle regulation by inducing the expression of key genes for the progression from G1 to S phase, such as CCND1 (PubMed:17344214). Mediates the effects of LEP on melanocortin production, body energy homeostasis and lactation (By similarity). May play an apoptotic role by transctivating BIRC5 expression under LEP activation (PubMed:18242580). Cytoplasmic STAT3 represses macroautophagy by inhibiting EIF2AK2/PKR activity (PubMed:23084476).

Uniprot:	P40763
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Signal transducer and activator of transcription 3
Sub Unit:	(Microbial infection) Interacts with HCV core protein.
Research Area:	Cancer
Subcellular Location:	Cytoplasm Nucleus Shuttles between the nucleus and the cytoplasm. Translocated into the nucleus upon tyrosine phosphorylation and dimerization, in response to signaling by activated FGFR1, FGFR2, FGFR3 or FGFR4. Constitutive nuclear presence is independent of tyrosine phosphorylation. Predominantly present in the cytoplasm without stimuli. Upon leukemia inhibitory factor (LIF) stimulation, accumulates in the nucleus. The complex composed of BART and ARL2 plays an important role in the nuclear translocation and retention of STAT3. Identified in a complex with LYN and PAG1.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	STAT3: transcription factor of the STAT family. Phosphorylated and activated by receptor-associated kinases downstream of many cytokines and growth-factor receptors. Constitutively active in a number of human tumors. 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:Motility/polarity/chemotaxis; Nuclear receptor co-regulator; DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 17q21.31 Cellular Component: nucleoplasm; cytoplasm; mitochondrial inner membrane; plasma membrane; nucleus; cytosol Molecular Function:protein dimerization activity; ligand-dependent nuclear receptor activity; protein binding; signal transducer activity; DNA binding; sequence-specific DNA binding; protein phosphatase binding; transcription factor binding; protein kinase binding; transcription factor activity; glucocorticoid receptor binding; CCR5 chemokine receptor binding Biological Process: transcription from RNA polymerase II promoter; viral reproduction; nerve growth factor receptor signaling pathway; somatic stem cell maintenance; positive regulation of transcription, DNA-dependent; radial glial cell differentiation; negative regulation of transcription from RNA polymerase II promoter; thermoregulation; signal transduction; glucose homeostasis; response to estradiol stimulus; negative regulation of cell proliferation; regulation of transcription, DNA-dependent; astrocyte differentiation; protein import into nucleus; acute-phase response; negative regulation of glycolysis; positive regulation of Notch signaling pathway; response to drug; nervous system development; intracellular receptor-mediated signaling pathway; eating behavior; cytokine and chemokine mediated signaling pathway; regulation of multicellular organism growth; JAK-STAT cascade; cellular response to hormone stimulus; regulation of transcription from RNA polymerase II promoter; cell proliferation; response to ethanol; sexual reproduction; positive regulation of transcription from RNA polymerase II promoter; cell motility; eye photoreceptor cell differentiation; phosphorylation Disease: Hyper-ige Recurrent Infection Syndrome, Autosomal Dominant; Autoimmune Disease, Multisystem, Infantile-onset
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 is activated through phosphorylation in response to various cytokines and growth factors including IFNs, EGF, IL5, IL6, HGF, LIF and BMP2. This protein mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. The small GTPase Rac1 has been shown to bind and regulate the activity of this protein. PIAS3 protein is a specific inhibitor of this protein. Three alternatively spliced transcript variants encoding distinct isoforms have been described. [provided by RefSeq, Jul 2008]
UniProt Code:	P40763
NCBI GenInfo Identifier:	48429227
NCBI Gene ID:	6774
NCBI Accession:	P40763.2
UniProt Secondary Accession:	P40763,O14916, Q9BW54, A8K7B8, K7ENL3,
UniProt Related Accession:	P40763
Molecular Weight:	770
NCBI Full Name:	Signal transducer and activator of transcription 3
NCBI Synonym Full Names:	signal transducer and activator of transcription 3 (acute-phase response factor)
NCBI Official Symbol:	STAT3
NCBI Official Synonym Symbols:	APRF; HIES; ADMIO
NCBI Protein Information:	signal transducer and activator of transcription 3; DNA-binding protein APRF; acute-phase response factor
UniProt Protein Name:	Signal transducer and activator of transcription 3
UniProt Synonym Protein Names:	Acute-phase response factor
Protein Family:	Signal transducer and activator of transcription
UniProt Gene Name:	STAT3
UniProt Entry Name:	STAT3_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 STAT3 ELISA Kit	Anti-STAT3 Antibody (CAB11216)
Human STAT3 (Signal Transducer and Activator of Transcription 3) CLIA Kit (HUES01131)	Anti-STAT3 Antibody (CAB1192)[KO Validated]
STAT3 Transcription Factor Activity Assay	Anti-STAT3 Antibody (CAB15729)
STAT3 (Phospho-Tyr705) Colorimetric Cell-Based ELISA Kit	Anti-STAT3 Antibody (CAB16975)
	Anti-STAT3 Antibody [KO Validated] (CAB19566)