Human STAT5B ELISA Kit (HUEB2649)- High Sensitivity

Product Name:	Human Signal transducer and activator of transcription 5B (STAT5B) ELISA Kit
SKU:	HUEB2649
Size:	96T
Target:	Human Signal transducer and activator of transcription 5B (STAT5B)
Synonyms:	Signal transducer and activator of transcription 5B, STAT5B
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.78-50ng/mL
Sensitivity:	0.34ng/mL

Intra CV:

4.9%

Inter CV:

9.0%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	84-93%	106-116%	91-100%	89-99%
EDTA Plasma(N=5)	102-114%	86-98%	94-104%	101-110%
Heparin Plasma(N=5)	102-111%	94-104%	107-117%	103-113%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	100	94-106
Plasma	102	96-108

Function:

Carries out a dual function: signal transduction and activation of transcription. Mediates cellular responses to the cytokine KITLG/SCF and other growth factors. Binds to the GAS element and activates PRL-induced transcription (PubMed:11773439). Positively regulates hematopoietic/erythroid differentiation (PubMed:20702587).

Uniprot:	P51692
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Signal transducer and activator of transcription 5B
Sub Unit:	Forms a homodimer or a heterodimer with a related family member. Binds NR3C1 (By similarity). Interacts with NCOA1 (PubMed:12954634). Interacts with NMI (PubMed:9989503). Interacts with SOCS7 (PubMed:15677474). Interacts (via SH2 domain) with INSR (PubMed:9428692). Interacts with CPEB3; this inhibits STAT5B-mediated transcriptional activation (PubMed:20639532).
Research Area:	Signal Transduction
Subcellular Location:	Cytoplasm Nucleus Translocated into the nucleus in response to phosphorylation.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	STAT5B: transcription factor of the STAT family. Phosphorylated and activated by receptor-associated kinases triggered by cytokines including IL2, IL3, GM-CSF, and various growth hormones. It has been shown to be involved in diverse biological processes, such as TCR signaling, apoptosis, adult mammary gland development, and sexual dimorphism of liver gene expression. Forms homo- or heterodimers that translocate into the nucleus where they regulate transcription.
UniProt Protein Details:	Protein type:Oncoprotein; Transcription factor; DNA-binding Chromosomal Location of Human Ortholog: 17q11.2 Cellular Component: cytoplasm; cytosol; nucleoplasm; nucleus Molecular Function:chromatin binding; glucocorticoid receptor binding; protein binding; protein dimerization activity; protein phosphatase binding; signal transducer activity; transcription factor activity Biological Process: 2-oxoglutarate metabolic process; acute-phase response; allantoin metabolic process; cellular response to hormone stimulus; citrate metabolic process; creatine metabolic process; creatinine metabolic process; development of secondary female sexual characteristics; development of secondary male sexual characteristics; fatty acid metabolic process; female pregnancy; isoleucine metabolic process; JAK-STAT cascade; lactation; liver development; luteinization; natural killer cell differentiation; negative regulation of apoptosis; negative regulation of erythrocyte differentiation; oxaloacetate metabolic process; Peyer's patch development; positive regulation of activated T cell proliferation; positive regulation of B cell differentiation; positive regulation of cell motility; positive regulation of gamma-delta T cell differentiation; positive regulation of inflammatory response; positive regulation of interleukin-2 biosynthetic process; positive regulation of mitotic cell cycle; positive regulation of multicellular organism growth; positive regulation of natural killer cell differentiation; positive regulation of natural killer cell mediated cytotoxicity; positive regulation of natural killer cell proliferation; positive regulation of smooth muscle cell proliferation; positive regulation of transcription from RNA polymerase II promoter; progesterone metabolic process; regulation of epithelial cell differentiation; regulation of multicellular organism growth; regulation of steroid metabolic process; regulation of transcription from RNA polymerase II promoter; response to estradiol stimulus; response to ethanol; response to hypoxia; response to lipopolysaccharide; sequestering of lipid; succinate metabolic process; T cell differentiation in the thymus; T cell homeostasis; taurine metabolic process; transcription from RNA polymerase II promoter; valine metabolic process Disease: Growth Hormone Insensitivity With Immunodeficiency
NCBI Summary:	The protein encoded by this gene is a member of the STAT family of transcription factors. 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 mediates the signal transduction triggered by various cell ligands, such as IL2, IL4, CSF1, and different growth hormones. It has been shown to be involved in diverse biological processes, such as TCR signaling, apoptosis, adult mammary gland development, and sexual dimorphism of liver gene expression. This gene was found to fuse to retinoic acid receptor-alpha (RARA) gene in a small subset of acute promyelocytic leukemias (APLL). The dysregulation of the signaling pathways mediated by this protein may be the cause of the APLL. [provided by RefSeq, Jul 2008]
UniProt Code:	P51692
NCBI GenInfo Identifier:	41019536
NCBI Gene ID:	6777
NCBI Accession:	P51692.2
UniProt Secondary Accession:	P51692,Q8WWS8,
UniProt Related Accession:	P51692
Molecular Weight:	89,866 Da
NCBI Full Name:	Signal transducer and activator of transcription 5B
NCBI Synonym Full Names:	signal transducer and activator of transcription 5B
NCBI Official Symbol:	STAT5B
NCBI Official Synonym Symbols:	STAT5
NCBI Protein Information:	signal transducer and activator of transcription 5B
UniProt Protein Name:	Signal transducer and activator of transcription 5B
UniProt Gene Name:	STAT5B
UniProt Entry Name:	STA5B_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 STAT5b ELISA Kit	Anti-STAT5B Antibody (CAB0275)
STAT5B (Phospho-Ser731) Transcription Factor Activity Assay	Anti-STAT5B Antibody (CAB12356)
STAT5B Transcription Factor Activity Assay	Anti-STAT5B Antibody [KO Validated] (CAB19567)
	TBP Antibody (PACO05870)
	STAT5A Antibody (PACO12514)