MYB (Phospho-Ser12) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB01302
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Reactivity:
Mouse
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:MYB (Phospho-Ser12) Colorimetric Cell-Based ELISA
Product Code:CBCAB01302
ELISA Type:Cell-Based
Target:MYB (Phospho-Ser12)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The MYB (Phospho-Ser12) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MYB protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated MYB in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on MYB phosphorylation.

Qualitative determination of MYB (Phospho-Ser12) concentration is achieved by an indirect ELISA format. In essence, MYB (Phospho-Ser12) is captured by MYB (Phospho-Ser12)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 4602, UniProt ID: P10242, OMIM: 189990, Unigene: Hs.654446
Gene Symbol:MYB
Sub Type:Phospho
UniProt Protein Function:Myb: Transcriptional activator; DNA-binding protein that specifically recognize the sequence 5'-YAAC[GT]G-3'. Plays an important role in the control of proliferation and differentiation of hematopoietic progenitor cells. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:DNA-binding; Transcription factor; Oncoprotein

Chromosomal Location of Human Ortholog: 6q22-q23

Molecular Function:protein binding

Biological Process: chromatin remodeling; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of histone H3-K4 methylation; positive regulation of histone H3-K9 methylation; positive regulation of T-helper cell differentiation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent

NCBI Summary:This gene encodes a protein with three HTH DNA-binding domains that functions as a transcription regulator. This protein plays an essential role in the regulation of hematopoiesis. This gene may be aberrently expressed or rearranged or undergo translocation in leukemias and lymphomas, and is considered to be an oncogene. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2016]
UniProt Code:P10242
NCBI GenInfo Identifier:2815504
NCBI Gene ID:4602
NCBI Accession:P10242.2
UniProt Secondary Accession:P10242,P78391, P78392, P78525, P78526, Q14023, Q14024 E9PI07, E9PLZ5, E9PNA4, E9PNL6, E9PRS2,
UniProt Related Accession:P10242
Molecular Weight:37,507 Da
NCBI Full Name:Transcriptional activator Myb
NCBI Synonym Full Names:MYB proto-oncogene, transcription factor
NCBI Official Symbol:MYB  
NCBI Official Synonym Symbols:efg; Cmyb; c-myb; c-myb_CDS  
NCBI Protein Information:transcriptional activator Myb
UniProt Protein Name:Transcriptional activator Myb
UniProt Synonym Protein Names:Proto-oncogene c-Myb
Protein Family:Myb protein
UniProt Gene Name:MYB  
UniProt Entry Name:MYB_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-MYB (Phospho-Ser12) Antibody, Anti-MYB 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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