Human PIAS3 ELISA Kit (HUEB1805)- High Sensitivity

Product Name:	Human E3 SUMO-protein ligase PIAS3 (PIAS3) ELISA Kit
SKU:	HUEB1805
Size:	96T
Target:	Human E3 SUMO-protein ligase PIAS3 (PIAS3)
Synonyms:	E3 SUMO-protein transferase PIAS3, Protein inhibitor of activated STAT protein 3
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	78-5000pg/mL
Sensitivity:	41.2pg/mL

Intra CV:

4.9%

Inter CV:

6.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	116-126%	108-120%	87-99%	92-101%
EDTA Plasma(N=5)	94-103%	93-103%	109-120%	89-101%
Heparin Plasma(N=5)	108-117%	101-111%	81-90%	105-114%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	101	95-107
Plasma	103	97-109

Function:

Functions as an E3-type small ubiquitin-like modifier (SUMO) ligase, stabilizing the interaction between UBE2I and the substrate, and as a SUMO-tethering factor. Plays a crucial role as a transcriptional coregulation in various cellular pathways, including the STAT pathway and the steroid hormone signaling pathway. Involved in regulating STAT3 signaling via inhibiting STAT3 DNA-binding and suppressing cell growth. Enhances the sumoylation of MTA1 and may participate in its paralog-selective sumoylation (PubMed:21965678, PubMed:9388184). Sumoylates CCAR2 which promotes its interaction with SIRT1 (PubMed:25406032). Diminishes the sumoylation of ZFHX3 by preventing the colocalization of ZFHX3 with SUMO1 in the nucleus (PubMed:24651376).

Uniprot:	Q9Y6X2
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human E3 SUMO-protein ligase PIAS3
Sub Unit:	Monomer (By similarity). Binds SUMO1 and UBE2I. Interacts with BCL11A, HMGA2, IRF1, MITF and NCOA2. Interacts with STAT5; the interaction occurs on stimulation by PRL. Interacts with GFI1; the interaction relieves the inhibitory effect of PIAS3 on STAT3-mediated transcriptional activity (By similarity). Interacts with AR, PLAG1 and ZFHX3. Interacts with STAT3; the interaction occurs on stimulation by IL6, CNTF or OSM and inhibits the DNA binding activity of STAT3. Interacts with MTA1. Interacts with CCAR2 (via N-terminus). Interacts with TRIM8 (PubMed:20516148).
Subcellular Location:	Cytoplasm Nucleus Nucleus speckle Colocalizes with MITF in the nucleus. Colocalizes with GFI1 in nuclear dots. Colocalizes with SUMO1 in nuclear granules.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	PIAS3: Functions as an E3-type small ubiquitin-like modifier (SUMO) ligase, stabilizing the interaction between UBE2I and the substrate, and as a SUMO-tethering factor. Plays a crucial role as a transcriptional coregulation in various cellular pathways, including the STAT pathway and the steroid hormone signaling pathway. Involved in regulating STAT3 signaling via inhibiting STAT3 DNA-binding and suppressing cell growth. Monomer. Binds SUMO1 and UBE2I. Interacts with BCL11A, HMGA2, IRF1, MITF and NCOA2. Interacts with STAT5; the interaction occurs on stimulation by PRL. Interacts with GFI1; the interaction relieves the inhibitory effect of PIAS3 on STAT3- mediated transcriptional activity. Interacts with AR, PLAG1 and ZFHX3. Interacts with STAT3; the interaction occurs on stimulation by IL6, CNTF or OSM and inhibits the DNA binding activity of STAT3. By dihydrotestosterone (DHT) in prostate cancer cells. Isoform 1 is expressed in most tissues except thymus and small intestine. Isoform 3 is expressed only in brain, heart, thymus, muscle, lung, testis, lactating breast and embryonic stem cells. Belongs to the PIAS family.
UniProt Protein Details:	Protein type:EC 6.3.2.-; SUMO conjugating system; Nuclear receptor co-regulator Chromosomal Location of Human Ortholog: 1q21 Cellular Component: cytoplasm; dendrite; synapse; nucleus Molecular Function:protein C-terminus binding; protein binding; potassium channel regulator activity; enzyme binding; zinc ion binding; protein N-terminus binding; SUMO ligase activity; ligase activity Biological Process: positive regulation of protein sumoylation; protein sumoylation; transcription, DNA-dependent; regulation of transcription, DNA-dependent; response to hormone stimulus; positive regulation of membrane potential
NCBI Summary:	This gene encodes a member of the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] family of transcriptional modulators. The protein functions as a SUMO (small ubiquitin-like modifier)-E3 ligase which catalyzes the covalent attachment of a SUMO protein to specific target substrates. It directly binds to several transcription factors and either blocks or enhances their activity. Alternatively spliced transcript variants of this gene have been identified, but the full-length nature of some of these variants has not been determined. [provided by RefSeq, Jul 2008]
UniProt Code:	Q9Y6X2
NCBI GenInfo Identifier:	56405390
NCBI Gene ID:	10401
NCBI Accession:	Q9Y6X2.2
UniProt Secondary Accession:	Q9Y6X2,Q9UFI3,
UniProt Related Accession:	Q9Y6X2
Molecular Weight:	628
NCBI Full Name:	E3 SUMO-protein ligase PIAS3
NCBI Synonym Full Names:	protein inhibitor of activated STAT, 3
NCBI Official Symbol:	PIAS3
NCBI Official Synonym Symbols:	ZMIZ5
NCBI Protein Information:	E3 SUMO-protein ligase PIAS3; zinc finger, MIZ-type containing 5; protein inhibitor of activated STAT protein 3
UniProt Protein Name:	E3 SUMO-protein ligase PIAS3
UniProt Synonym Protein Names:	Protein inhibitor of activated STAT protein 3
Protein Family:	E3 SUMO-protein ligase
UniProt Gene Name:	PIAS3
UniProt Entry Name:	PIAS3_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 PIAS3 (Protein Inhibitor of Activated STAT3) ELISA Kit (HUES02061)	Anti-PIAS3 Antibody (CAB13394)
PIAS3 Colorimetric Cell-Based ELISA Kit	Anti-PIAS3 Antibody (CAB7060)
	PIK3R2 Antibody (PACO06749)
	PIAS2 Antibody (PACO11231)
	PIAS2 Antibody (PACO23145)