CtBP1 (Phospho-Ser422)Colorimetric Cell-Based ELISA Kit (CBCAB01678)

Product Name:	CtBP1 (Phospho-Ser422)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01678
ELISA Type:	Cell-Based
Target:	CtBP1 (Phospho-Ser422)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The CtBP1 (Phospho-Ser422) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CtBP1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CtBP1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CtBP1 phosphorylation.

Qualitative determination of CtBP1 (Phospho-Ser422) concentration is achieved by an indirect ELISA format. In essence, CtBP1 (Phospho-Ser422) is captured by CtBP1 (Phospho-Ser422)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ 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: 1487, UniProt ID: Q13363, OMIM: 602618, Unigene: Hs.208597
Gene Symbol:	CTBP1
Sub Type:	Phospho

UniProt Protein Function:	CTBP1: Involved in controlling the equilibrium between tubular and stacked structures in the Golgi complex. Functions in brown adipose tissue (BAT) differentiation. Corepressor targeting diverse transcription regulators such as GLIS2. Has dehydrogenase activity. Homo- or heterodimer. Heterodimer with CTBP2. Interacts with PRDM16; the interaction represses white adipose tissue (WAT)- specific genes expression. Interacts with GLIS2, FOXP2, HDAC4, HDAC5, HDAC9 and ZNF217. Interacts with adenovirus E1A protein (via its C-terminus); the interaction disrupts the interaction of CTBP1 with RBBP8. Interacts with Epstein-Barr virus EBNA3 and EBNA6. Interacts with ELK3 (via its PXDLS motif). Interacts with RBBP8 (via its PXDLS motif); the interaction is disrupted by binding to adenovirus E1A. Interacts with FOXP1, HIPK2, PNN, NRIP1, MECOM, ZNF366, ZFHX1B and WIZ. Interaction with SATB1 (non-acetylated form); the interaction stabilizes its attachment to DNA and promotes transcription repression. Belongs to the D-isomer specific 2-hydroxyacid dehydrogenase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Oxidoreductase; Nuclear receptor co-regulator; EC 1.1.1.-; Transcription, coactivator/corepressor; Cell cycle regulation Chromosomal Location of Human Ortholog: 4p16 Cellular Component: nucleoplasm; transcription factor complex; transcriptional repressor complex; cytosol; nucleus Molecular Function:protein C-terminus binding; protein domain specific binding; protein binding; protein homodimerization activity; transcription factor binding; NAD binding; transcription factor activity; oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor Biological Process: negative regulation of cell proliferation; positive regulation of histone deacetylation; transcription, DNA-dependent; regulation of cell cycle; white fat cell differentiation; viral genome replication; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; Golgi organization and biogenesis; protein amino acid phosphorylation; negative regulation of histone acetylation Disease: Wolf-hirschhorn Syndrome
NCBI Summary:	This gene encodes a protein that binds to the C-terminus of adenovirus E1A proteins. This phosphoprotein is a transcriptional repressor and may play a role during cellular proliferation. This protein and the product of a second closely related gene, CTBP2, can dimerize. Both proteins can also interact with a polycomb group protein complex which participates in regulation of gene expression during development. Alternative splicing of transcripts from this gene results in multiple transcript variants. [provided by RefSeq, Jul 2008]
UniProt Code:	Q13363
NCBI GenInfo Identifier:	6014741
NCBI Gene ID:	1487
NCBI Accession:	Q13363.2
UniProt Secondary Accession:	Q13363,Q4W5N3, Q7Z2Q5,
UniProt Related Accession:	Q13363
Molecular Weight:	440
NCBI Full Name:	C-terminal-binding protein 1
NCBI Synonym Full Names:	C-terminal binding protein 1
NCBI Official Symbol:	CTBP1
NCBI Official Synonym Symbols:	BARS
NCBI Protein Information:	C-terminal-binding protein 1; brefeldin A-ribosylated substrate
UniProt Protein Name:	C-terminal-binding protein 1
UniProt Gene Name:	CTBP1
UniProt Entry Name:	CTBP1_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 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.

Step	Procedure
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-CtBP1 (Phospho-Ser422) Antibody, Anti-CtBP1 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.)