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STEA3 Colorimetric Cell-Based ELISA

SKU:
CBCAB01136
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Death
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

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STEA3 Colorimetric Cell-Based ELISA

The STEA3 Colorimetric Cell-Based ELISA kit from Assay Genie is specifically designed for the accurate detection of angiogenin levels in various biological samples, including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for researchers in various fields.Angiogenin is a key protein involved in angiogenesis, playing a crucial role in promoting blood vessel formation and cell proliferation.

Its involvement in conditions such as cancer, cardiovascular diseases, and neurodegenerative disorders makes it a valuable biomarker for studying these diseases and potential therapeutic interventions.With the STEA3 Colorimetric Cell-Based ELISA kit, researchers can confidently measure angiogenin levels in their samples, providing essential insights into disease mechanisms and potential treatment options. Trust Assay Genie for high-quality ELISA kits for your research needs.

Product Name:STEA3 Colorimetric Cell-Based ELISA
Product Code:CBCAB01136
ELISA Type:Cell-Based
Target:STEA3
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The STEA3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect STEA3 protein expression profile in cells. The kit can be used for measuring the relative amounts of STEA3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on STEA3.

Qualitative determination of STEA3 concentration is achieved by an indirect ELISA format. In essence, STEA3 is captured by STEA3-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: 55240, UniProt ID: Q658P3, OMIM: 609671, Unigene: Hs.567828/Hs.647822
Gene Symbol:STEA3
Sub Type:None
UniProt Protein Function:STEAP3: a multi-pass membrane protein induced directly by p53. Plays an important role in facilitating exosome secretion. Enhances secretion in the DNA damage-induced p53-dependent exosomal secretory pathway. Localizes to vesicular-like structures at the plasma membrane and around the nucleus. Highly expressed in hematopoietic tissues where it localizes to the specialized endosome. An endosomal ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells. Participates in erythroid iron homeostasis by reducing Fe(3+) to Fe(2+). Can also reduce of Cu(2+) to Cu(1+), suggesting that it participates in copper homeostasis. Uses NAD(+) as acceptor. Contains a highly-conserved tyrosine-based motif that has been shown to target transmembrane proteins, including LAMP-1, LAMP-2, the TfR, and SLC11A1 to endosomes, lysosomes, and lysosome-related organelles. Facilitates the exosomal secretion of proteins such as TCTP. Interacts with BNIP3L, MYT1 and TPT1. Four human isoforms are produced by alternative splicing. Isoform 4 is also known as pHyde II.
UniProt Protein Details:

Protein type:Membrane protein, integral; Membrane protein, multi-pass; EC 1.16.1.-; Transporter, iron; Transporter, ion channel; Oxidoreductase; Transporter

Chromosomal Location of Human Ortholog: 2q14.2

Cellular Component: multivesicular body; integral to membrane; plasma membrane; endosome membrane

Molecular Function:protein binding; metal ion binding; cupric reductase activity; ferric-chelate reductase activity

Biological Process: apoptosis; cellular iron ion homeostasis; protein secretion; transferrin transport; copper ion import; cell cycle; transmembrane transport

Disease: Anemia, Hypochromic Microcytic, With Iron Overload 2

NCBI Summary:This gene encodes a multipass membrane protein that functions as an iron transporter. The encoded protein can reduce both iron (Fe3+) and copper (Cu2+) cations. This protein may mediate downstream responses to p53, including promoting apoptosis. Deficiency in this gene can cause anemia. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Aug 2015]
UniProt Code:Q658P3
NCBI GenInfo Identifier:146325737
NCBI Gene ID:55240
NCBI Accession:Q658P3.2
UniProt Secondary Accession:Q658P3,Q4VBR2, Q4ZG36, Q53SQ8, Q7Z389, Q86SF6, Q8NEW6 Q8TDP3, Q8TF03, Q9NVB5, A8K6E3,
UniProt Related Accession:Q658P3
Molecular Weight:488
NCBI Full Name:Metalloreductase STEAP3
NCBI Synonym Full Names:STEAP family member 3, metalloreductase
NCBI Official Symbol:STEAP3  
NCBI Official Synonym Symbols:STMP3; TSAP6; AHMIO2; dudlin-2  
NCBI Protein Information:metalloreductase STEAP3; pHyde; hTSAP6; hpHyde; dudlin 2; dudulin 2; dudulin-2; 1010001D01Rik; tumor suppressor pHyde; six transmembrane prostate protein 3; tumor suppressor-activated pathway protein 6; six-transmembrane epithelial antigen of prostate 3
UniProt Protein Name:Metalloreductase STEAP3
UniProt Synonym Protein Names:Dudulin-2; Six-transmembrane epithelial antigen of prostate 3; Tumor suppressor-activated pathway protein 6; hTSAP6; pHyde; hpHyde
Protein Family:Metalloreductase
UniProt Gene Name:STEAP3  
UniProt Entry Name:STEA3_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-STEA3 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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