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MAK Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00739
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Developmental Biology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

MAK Colorimetric Cell-Based ELISA Kit

The MAK Colorimetric Cell-Based ELISA Kit from AssayGenie is a powerful tool for researchers looking to accurately detect angiogenin levels in human serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit provides reliable and reproducible results for a wide range of research applications.Angiogenin is a key protein involved in angiogenesis, playing a crucial role in blood vessel formation and cell proliferation.

Its implications in conditions such as cancer, cardiovascular diseases, and neurodegenerative disorders make it a vital biomarker for studying these conditions and developing potential therapies.Trust the MAK Colorimetric Cell-Based ELISA Kit from AssayGenie to help you advance your research and uncover important insights into angiogenin levels and their role in various diseases.

Product Name:MAK Colorimetric Cell-Based ELISA
Product Code:CBCAB00739
ELISA Type:Cell-Based
Target:MAK
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of MAK concentration is achieved by an indirect ELISA format. In essence, MAK is captured by MAK-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: 4117, UniProt ID: P20794, OMIM: 154235, Unigene: Hs.446125
Gene Symbol:MAK
Sub Type:None
UniProt Protein Function:MAK: a protein kinase of the RCK family of kinases. It is expressed at high levels in the testis (primarily in germ cells), and in developing sensory epithelia. Localizes to the chromosomes during meiosis in spermatogenesis, specifically to the synaptonemal complex that exists while homologous chromosomes are paired.
UniProt Protein Details:

Protein type:Protein kinase, CMGC; EC 2.7.11.22; Nuclear receptor co-regulator; Kinase, protein; Protein kinase, Ser/Thr (non-receptor); CMGC group; RCK family

Chromosomal Location of Human Ortholog: 6p24

Cellular Component: centrosome; midbody; nucleus; photoreceptor inner segment; photoreceptor outer segment

Molecular Function:protein binding; transcription coactivator activity

Biological Process: photoreceptor cell maintenance; protein amino acid autophosphorylation; protein amino acid phosphorylation

Disease: Retinitis Pigmentosa 62

NCBI Summary:The product of this gene is a serine/threonine protein kinase related to kinases involved in cell cycle regulation. Studies of the mouse and rat homologs have localized the kinase to the chromosomes during meiosis in spermatogenesis, specifically to the synaptonemal complex that exists while homologous chromosomes are paired. Mutations in this gene have been associated with ciliary defects resulting in retinitis pigmentosa 62. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2016]
UniProt Code:P20794
NCBI GenInfo Identifier:13432166
NCBI Gene ID:4117
NCBI Accession:P20794.2
UniProt Secondary Accession:P20794,Q547D0, Q9NUH7, F1T0K6, G1FL29,
UniProt Related Accession:P20794
Molecular Weight:66,345 Da
NCBI Full Name:Serine/threonine-protein kinase MAK
NCBI Synonym Full Names:male germ cell associated kinase
NCBI Official Symbol:MAK  
NCBI Official Synonym Symbols:RP62  
NCBI Protein Information:serine/threonine-protein kinase MAK
UniProt Protein Name:Serine/threonine-protein kinase MAK
UniProt Synonym Protein Names:Male germ cell-associated kinase
Protein Family:Makatoxin
UniProt Gene Name:MAK  
UniProt Entry Name:MAK_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-MAK 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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