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MOBKL2B Colorimetric Cell-Based ELISA

SKU:
CBCAB01144
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheet

MOBKL2B Colorimetric Cell-Based ELISA

The MOBKL2B Colorimetric Cell-Based ELISA Kit is specifically designed to detect MOBKL2B levels in cell lysates and culture supernatants. This kit offers high sensitivity and specificity, providing accurate and reproducible results for a variety of research applications.MOBKL2B, also known as MOB1B, is a key regulator of cell growth and division, playing a crucial role in cell cycle progression and cell proliferation. By monitoring MOBKL2B levels, researchers can gain insights into various cellular processes and pathways, such as signal transduction and cell differentiation.

Whether studying cancer biology, cell signaling, or drug discovery, the MOBKL2B Colorimetric Cell-Based ELISA Kit offers a powerful tool for investigating the role of MOBKL2B in cellular physiology and disease progression. Get reliable and comprehensive data with this advanced ELISA kit from AssayGenie.

Product Name:MOBKL2B Colorimetric Cell-Based ELISA
Product Code:CBCAB01144
ELISA Type:Cell-Based
Target:MOBKL2B
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The MOBKL2B Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MOBKL2B protein expression profile in cells. The kit can be used for measuring the relative amounts of MOBKL2B in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MOBKL2B.

Qualitative determination of MOBKL2B concentration is achieved by an indirect ELISA format. In essence, MOBKL2B is captured by MOBKL2B-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: 79817, UniProt ID: Q86TA1, OMIM: 0, Unigene: Hs.369022/Hs.690014/Hs.551550
Gene Symbol:MOBKL2B
Sub Type:None
UniProt Protein Function:MOBKL2B: May regulate the activity of kinases. Belongs to the MOB1/phocein family.
UniProt Protein Details:

Chromosomal Location of Human Ortholog: 9p21.2

Molecular Function:protein binding

NCBI Summary:The protein encoded by this gene shares similarity with the yeast Mob1 protein. Yeast Mob1 binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. This gene is located on the opposite strand as the interferon kappa precursor (IFNK) gene. [provided by RefSeq, Jul 2008]
UniProt Code:Q86TA1
NCBI GenInfo Identifier:56749334
NCBI Gene ID:79817
NCBI Accession:Q86TA1.2
UniProt Secondary Accession:Q86TA1,Q8NEB4, Q9H8V4,
UniProt Related Accession:Q86TA1
Molecular Weight:25,464 Da
NCBI Full Name:MOB kinase activator 3B
NCBI Synonym Full Names:MOB kinase activator 3B
NCBI Official Symbol:MOB3B  
NCBI Official Synonym Symbols:MOB1D; C9orf35; MOBKL2B  
NCBI Protein Information:MOB kinase activator 3B
UniProt Protein Name:MOB kinase activator 3B
UniProt Synonym Protein Names:Mob1 homolog 2b; Mps one binder kinase activator-like 2B
Protein Family:MOB kinase activator
UniProt Gene Name:MOB3B  
UniProt Entry Name:MOB3B_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-MOBKL2B 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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