MARCKS Colorimetric Cell-Based ELISA Kit (CBCAB00745)

Product Name:	MARCKS Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00745
ELISA Type:	Cell-Based
Target:	MARCKS
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of MARCKS concentration is achieved by an indirect ELISA format. In essence, MARCKS is captured by MARCKS-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: 4082, UniProt ID: P29966, OMIM: 177061, Unigene: Hs.519909/Hs.712721
Gene Symbol:	MARCKS
Sub Type:	None

UniProt Protein Function:	MARCKS: the most prominent cellular substrate of protein kinase C. Binds calmodulin, actin, and synapsin. A filamentous actin cross-linking protein. Critical for normal brain development. Phosphorylation by PKC displaces it from the plasma membrane, inhibiting F-actin cross-linking activity. Plays a role in growth cone adhesion: its nonphosphorylated form stabilizes integrin-mediated adhesions, and its phosphorylation-triggered release from adhesions causes localized growth cone detachment. Highly expressed in immature T and B lymphocytes: its expression is highest in CD4-8- T cells and B-cell blasts generated by antigen receptor signaling. Its expression and phosphorylation are closely linked to microglial activation processes involved in pathological conditions such as neuroinflammation and neurodegeneration.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Actin-binding Chromosomal Location of Human Ortholog: 6q22.2 Cellular Component: actin cytoskeleton; cell cortex; centrosome; cytosol; dendritic spine; focal adhesion; germinal vesicle; growth cone; lysosome; microtubule; nerve terminal; outer dense fiber; plasma membrane Molecular Function:actin filament binding; calmodulin binding; phosphatidylserine binding; protein kinase C binding Biological Process: actin filament organization; brain development; cell-substrate adhesion; energy reserve metabolic process; intracellular protein transport; phospholipase D activation; regulation of insulin secretion
NCBI Summary:	The protein encoded by this gene is a substrate for protein kinase C. It is localized to the plasma membrane and is an actin filament crosslinking protein. Phosphorylation by protein kinase C or binding to calcium-calmodulin inhibits its association with actin and with the plasma membrane, leading to its presence in the cytoplasm. The protein is thought to be involved in cell motility, phagocytosis, membrane trafficking and mitogenesis. [provided by RefSeq, Jul 2008]
UniProt Code:	P29966
NCBI GenInfo Identifier:	76803798
NCBI Gene ID:	4082
NCBI Accession:	P29966.4
UniProt Secondary Accession:	P29966,Q2LA83, Q5TDB7, E1P560,
UniProt Related Accession:	P29966
Molecular Weight:
NCBI Full Name:	Myristoylated alanine-rich C-kinase substrate
NCBI Synonym Full Names:	myristoylated alanine rich protein kinase C substrate
NCBI Official Symbol:	MARCKS
NCBI Official Synonym Symbols:	MACS; 80K-L; PKCSL; PRKCSL
NCBI Protein Information:	myristoylated alanine-rich C-kinase substrate
UniProt Protein Name:	Myristoylated alanine-rich C-kinase substrate
UniProt Synonym Protein Names:	Protein kinase C substrate, 80 kDa protein, light chain; 80K-L protein; PKCSL
Protein Family:	MARCKS-related protein
UniProt Gene Name:	MARCKS
UniProt Entry Name:	MARCS_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-MARCKS 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.)