Cell Cycle ELISA Kits

MAEA Colorimetric Cell-Based ELISA

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SKU:
CBCAB01142
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Cycle
Reactivity:
Human
Reactivity:
Mouse
Reactivity:
Rat
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:MAEA Colorimetric Cell-Based ELISA
Product Code:CBCAB01142
ELISA Type:Cell-Based
Target:MAEA
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of MAEA concentration is achieved by an indirect ELISA format. In essence, MAEA is captured by MAEA-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: 10296, UniProt ID: Q7L5Y9, OMIM: 606801, Unigene: Hs.139896
Gene Symbol:MAEA
Sub Type:None
UniProt Protein Function:MAEA: Plays a role in erythroblast enucleation and in the development of the mature macrophages. Mediates the attachment of erythroid cell to mature macrophages, in correlation with the presence of MAEA at cell surface of mature macrophages; This MAEA- mediated contact inhibits erythroid cells apoptosis. Participates to erythroblastic island formation, which is the functional unit of definitive erythropoiesis. Associates with F-actin to regulate actin distribution in erythroblasts and macrophages. May contribute to nuclear architecture and cells division events. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Cell adhesion; Actin-binding; Cell development/differentiation

Chromosomal Location of Human Ortholog: 4p16.3

Cellular Component: contractile ring; cytoplasm; cytoskeleton; integral to plasma membrane; nuclear matrix; nucleus; spindle

Molecular Function:actin binding

Biological Process: cell adhesion; negative regulation of myeloid cell apoptosis

NCBI Summary:This gene encodes a protein that mediates the attachment of erythroblasts to macrophages. This attachment promotes terminal maturation and enucleation of erythroblasts, presumably by suppressing apoptosis. The encoded protein is an integral membrane protein with the N-terminus on the extracellular side and the C-terminus on the cytoplasmic side of the cell. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2014]
UniProt Code:Q7L5Y9
NCBI GenInfo Identifier:74754297
NCBI Gene ID:10296
NCBI Accession:Q7L5Y9.1
UniProt Secondary Accession:Q7L5Y9,O95285, Q5JB54, Q6ZRD6, Q9BQ11, Q9H9V6, Q9H9Z4 Q9NW84,
UniProt Related Accession:Q7L5Y9
Molecular Weight:26,802 Da
NCBI Full Name:Macrophage erythroblast attacher
NCBI Synonym Full Names:macrophage erythroblast attacher
NCBI Official Symbol:MAEA  
NCBI Official Synonym Symbols:EMP; EMLP; GID9; PIG5; HLC-10  
NCBI Protein Information:macrophage erythroblast attacher
UniProt Protein Name:Macrophage erythroblast attacher
UniProt Synonym Protein Names:Cell proliferation-inducing gene 5 protein; Erythroblast macrophage protein; Human lung cancer oncogene 10 protein; HLC-10
Protein Family:Macrophage erythroblast attacher
UniProt Gene Name:MAEA  
UniProt Entry Name:MAEA_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-MAEA 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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