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PIAS1 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00812
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

PIAS1 Colorimetric Cell-Based ELISA Kit

The PIAS1 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the accurate measurement of PIAS1 levels in cell lysates and culture supernatants. This kit offers high sensitivity and specificity, providing researchers with reliable and reproducible results that are essential for studying the role of PIAS1 in various biological processes.PIAS1, also known as protein inhibitor of activated STAT 1, is a critical regulator of the JAK/STAT signaling pathway and is involved in a wide range of cellular functions, including immune response, apoptosis, and cell proliferation.

Aberrant PIAS1 expression has been linked to numerous diseases, including cancer and inflammatory disorders, making it a valuable target for therapeutic interventions and biomarker development.With the PIAS1 Colorimetric Cell-Based ELISA Kit, researchers can accurately quantify PIAS1 levels in cell samples, enabling them to better understand its role in disease pathogenesis and identify potential therapeutic targets. This innovative assay is a valuable tool for any research laboratory studying the complex interplay of PIAS1 in cellular signaling pathways.

Product Name:PIAS1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00812
ELISA Type:Cell-Based
Target:PIAS1
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The PIAS1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PIAS1 protein expression profile in cells. The kit can be used for measuring the relative amounts of PIAS1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on PIAS1.

Qualitative determination of PIAS1 concentration is achieved by an indirect ELISA format. In essence, PIAS1 is captured by PIAS1-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 8554, UniProt ID: O75925, OMIM: 603566, Unigene: Hs.162458
Gene Symbol:PIAS1
Sub Type:None
UniProt Protein Function:PIAS1: 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, the p53 pathway and the steroid hormone signaling pathway. In vitro, binds A/T-rich DNA. The effects of this transcriptional coregulation, transactivation or silencing, may vary depending upon the biological context. Together with PRMT1, may repress STAT1 transcriptional activity, in the late phase of interferon gamma (IFN-gamma) signaling. Interacts with NCOA2 and AR. Interacts with NR2C1; the interaction promotes its sumoylation. Interacts with DDX21, CSRP2, AXIN1, JUN, UBE2I, SUMO1, SATB2, PLAG1, TP53 and STAT1 (dimer), following IFNA1-stimulation. Interacts with SP3 (preferentially when SUMO-modified). Interacts with KLF8; the interaction results in SUMO ligation and repression of KLF8 transcriptional activity and of its cell cycle progression into G(1) phase. Interacts with STAT1. Interacts with CHUK/IKKA; this interaction induces PIAS1 phosphorylation. Interacts with PTK2/FAK1; the interaction promotes its sumoylation. Interacts with DDX5. Expressed in numerous tissues with highest level in testis. Belongs to the PIAS family.
UniProt Protein Details:

Protein type:EC 6.3.2.-; SUMO conjugating system; Nuclear receptor co-regulator; Transcription, coactivator/corepressor

Chromosomal Location of Human Ortholog: 15q

Cellular Component: nucleoplasm; nucleus; PML body

Molecular Function:enzyme binding; protein binding; SUMO ligase activity; transcription corepressor activity

Biological Process: JAK-STAT cascade; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; positive regulation of protein sumoylation; protein sumoylation; regulation of cell proliferation

NCBI Summary:This gene encodes a member of the protein inhibitor of activated STAT (PIAS) family. PIAS proteins function as SUMO E3 ligases and play important roles in many cellular processes by mediating the sumoylation of target proteins. This protein plays a central role as a transcriptional coregulator of numerous cellular pathways includign the STAT1 and nuclear factor kappaB pathways. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Mar 2016]
UniProt Code:O75925
NCBI GenInfo Identifier:20138891
NCBI Gene ID:8554
NCBI Accession:O75925.2
UniProt Secondary Accession:O75925,Q147X4, Q99751, Q9UN02, B2RB67, B3KSY9, C5J4B4
UniProt Related Accession:O75925
Molecular Weight:36,204 Da
NCBI Full Name:E3 SUMO-protein ligase PIAS1
NCBI Synonym Full Names:protein inhibitor of activated STAT 1
NCBI Official Symbol:PIAS1  
NCBI Official Synonym Symbols:GBP; ZMIZ3; DDXBP1; GU/RH-II  
NCBI Protein Information:E3 SUMO-protein ligase PIAS1
UniProt Protein Name:E3 SUMO-protein ligase PIAS1
UniProt Synonym Protein Names:DEAD/H box-binding protein 1; Gu-binding protein; GBP; Protein inhibitor of activated STAT protein 1; RNA helicase II-binding protein
Protein Family:E3 SUMO-protein ligase
UniProt Gene Name:PIAS1  
UniProt Entry Name:PIAS1_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

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

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies (Anti-PIAS1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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