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MAST3 Colorimetric Cell-Based ELISA

SKU:
CBCAB00937
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

MAST3 Colorimetric Cell-Based ELISA

The MAST3 Colorimetric Cell-Based ELISA is a cutting-edge assay kit designed for the accurate detection of Mast/stem cell growth factor receptor (MAST3) levels in cell lysates and tissue extracts. This kit offers high sensitivity and specificity, providing reliable and reproducible results for a variety of research applications.MAST3 is a critical protein that plays a key role in regulating cell growth, differentiation, and survival. It is involved in various cellular processes, including cell migration, adhesion, and signaling pathways.

Dysregulation of MAST3 has been linked to numerous diseases and disorders, making it a valuable biomarker for studying and developing potential therapies.With the MAST3 Colorimetric Cell-Based ELISA Kit, researchers can accurately measure MAST3 levels in their samples, allowing for a better understanding of its role in disease progression and potential therapeutic targets. This kit is a valuable tool for advancing research in fields such as cancer biology, cell biology, and drug discovery.

Product Name:MAST3 Colorimetric Cell-Based ELISA
Product Code:CBCAB00937
ELISA Type:Cell-Based
Target:MAST3
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The MAST3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MAST3 protein expression profile in cells. The kit can be used for measuring the relative amounts of MAST3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MAST3.

Qualitative determination of MAST3 concentration is achieved by an indirect ELISA format. In essence, MAST3 is captured by MAST3-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: 23031, UniProt ID: O60307, OMIM: 0, Unigene: Hs.466184
Gene Symbol:MAST3
Sub Type:None
UniProt Protein Function:MAST3: Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family.
UniProt Protein Details:

Protein type:EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, AGC; AGC group; MAST family

Chromosomal Location of Human Ortholog: 19p13.11

Cellular Component: intracellular

Molecular Function:ATP binding; magnesium ion binding; protein binding; protein serine/threonine kinase activity

Biological Process: cytoskeleton organization and biogenesis; peptidyl-serine phosphorylation

UniProt Code:O60307
NCBI GenInfo Identifier:82592942
NCBI Gene ID:23031
NCBI Accession:O60307.2
UniProt Related Accession:O60307
Molecular Weight:143,137 Da
NCBI Full Name:Microtubule-associated serine/threonine-protein kinase 3
NCBI Synonym Full Names:microtubule associated serine/threonine kinase 3
NCBI Official Symbol:MAST3  
NCBI Protein Information:microtubule-associated serine/threonine-protein kinase 3
UniProt Protein Name:Microtubule-associated serine/threonine-protein kinase 3
Protein Family:Microtubule-associated serine/threonine-protein kinase
UniProt Gene Name:MAST3  
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-MAST3 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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