SMC1 (Phospho-Ser966)Colorimetric Cell-Based ELISA Kit (CBCAB00237)

Product Name:	SMC1 (Phospho-Ser966)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00237
ELISA Type:	Cell-Based
Target:	SMC1 (Phospho-Ser966)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The SMC1 (Phospho-Ser966) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SMC1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SMC1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SMC1 phosphorylation.

Qualitative determination of SMC1 (Phospho-Ser966) concentration is achieved by an indirect ELISA format. In essence, SMC1 (Phospho-Ser966) is captured by SMC1 (Phospho-Ser966)-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: 8243, UniProt ID: Q14683, OMIM: 300040/300590, Unigene: Hs.211602
Gene Symbol:	SMC1A
Sub Type:	Phospho

UniProt Protein Function:	Involved in chromosome cohesion during cell cycle and in DNA repair. Central component of cohesin complex. The cohesin complex is required for the cohesion of sister chromatids after DNA replication. The cohesin complex apparently forms a large proteinaceous ring within which sister chromatids can be trapped. At anaphase, the complex is cleaved and dissociates from chromatin, allowing sister chromatids to segregate. The cohesin complex may also play a role in spindle pole assembly during mitosis. Involved in DNA repair via its interaction with BRCA1 and its related phosphorylation by ATM, or via its phosphorylation by ATR. Works as a downstream effector both in the ATM/NBS1 branch and in the ATR/MSH2 branch of S-phase checkpoint.
NCBI Summary:	Proper cohesion of sister chromatids is a prerequisite for the correct segregation of chromosomes during cell division. The cohesin multiprotein complex is required for sister chromatid cohesion. This complex is composed partly of two structural maintenance of chromosomes (SMC) proteins, SMC3 and either SMC1B or the protein encoded by this gene. Most of the cohesin complexes dissociate from the chromosomes before mitosis, although those complexes at the kinetochore remain. Therefore, the encoded protein is thought to be an important part of functional kinetochores. In addition, this protein interacts with BRCA1 and is phosphorylated by ATM, indicating a potential role for this protein in DNA repair. This gene, which belongs to the SMC gene family, is located in an area of the X-chromosome that escapes X inactivation. Mutations in this gene result in Cornelia de Lange syndrome. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2013]
UniProt Code:	Q14683
NCBI GenInfo Identifier:	29336622
NCBI Gene ID:	8243
NCBI Accession:	Q14683.2
UniProt Secondary Accession:	Q14683,O14995, Q16351, Q2M228,
UniProt Related Accession:	Q14683
Molecular Weight:	155 kDa
NCBI Full Name:	Structural maintenance of chromosomes protein 1A
NCBI Synonym Full Names:	structural maintenance of chromosomes 1A
NCBI Official Symbol:	SMC1A
NCBI Official Synonym Symbols:	SMC1; SMCB; CDLS2; SB1.8; SMC1L1; DXS423E; SMC1alpha
NCBI Protein Information:	structural maintenance of chromosomes protein 1A
UniProt Protein Name:	Structural maintenance of chromosomes protein 1A
UniProt Synonym Protein Names:	Sb1.8
Protein Family:	Structural maintenance of chromosomes protein
UniProt Gene Name:	SMC1A

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-SMC1 (Phospho-Ser966) Antibody, Anti-SMC1 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.)