Filamin A Colorimetric Cell-Based ELISA Kit (CBCAB00658)

Product Name:	Filamin A Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00658
ELISA Type:	Cell-Based
Target:	Filamin A
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of Filamin A concentration is achieved by an indirect ELISA format. In essence, Filamin A is captured by Filamin A-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: 2316, UniProt ID: P21333, OMIM: 300017, Unigene: Hs.195464
Gene Symbol:	FLNA
Sub Type:	None

UniProt Protein Function:	FLNA: a ubiquitous cytoskeletal protein that promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. Plays an essential role in embryonic cell migration. Anchors various transmembrane proteins to the actin cytoskeleton and serves as a scaffold for a wide range of cytoplasmic signaling proteins. Interactions with filamin A may allow neuroblast migration from the ventricular zone into the cortical plate. Tethers cell surface-localized furin, modulates its rate of internalization and directs its intracellular trafficking.
UniProt Protein Details:	Protein type:Actin-binding; Motility/polarity/chemotaxis; Nuclear receptor co-regulator Chromosomal Location of Human Ortholog: Xq28 Cellular Component: cortical cytoskeleton; focal adhesion; extracellular region; actin filament; trans-Golgi network; cytosol; actin cytoskeleton; cell soma; membrane; perinuclear region of cytoplasm; cytoplasm; plasma membrane; dendritic shaft; nucleus Molecular Function:Rho GTPase binding; protein homodimerization activity; Rac GTPase binding; Ral GTPase binding; transcription factor binding; actin filament binding; small GTPase binding; signal transducer activity; protein binding; protein kinase C binding; mu-type opioid receptor binding; glycoprotein binding; SMAD binding Biological Process: actin crosslink formation; platelet activation; positive regulation of I-kappaB kinase/NF-kappaB cascade; protein stabilization; negative regulation of transcription factor activity; mRNA transcription from RNA polymerase II promoter; receptor clustering; dopamine receptor, adenylate cyclase inhibiting pathway; establishment of protein localization; platelet degranulation; early endosome to late endosome transport; actin cytoskeleton reorganization; positive regulation of transcription factor import into nucleus; cytoplasmic sequestering of protein; epithelial to mesenchymal transition; cilium biogenesis; negative regulation of protein catabolic process; blood coagulation Disease: Terminal Osseous Dysplasia; Fg Syndrome 2; Cardiac Valvular Dysplasia, X-linked; Frontometaphyseal Dysplasia; Melnick-needles Syndrome; Heterotopia, Periventricular, Ehlers-danlos Variant; Intestinal Pseudoobstruction, Neuronal, Chronic Idiopathic, X-linked; Heterotopia, Periventricular, X-linked Dominant; Otopalatodigital Syndrome, Type Ii; Otopalatodigital Syndrome, Type I
NCBI Summary:	The protein encoded by this gene is an actin-binding protein that crosslinks actin filaments and links actin filaments to membrane glycoproteins. The encoded protein is involved in remodeling the cytoskeleton to effect changes in cell shape and migration. This protein interacts with integrins, transmembrane receptor complexes, and second messengers. Defects in this gene are a cause of several syndromes, including periventricular nodular heterotopias (PVNH1, PVNH4), otopalatodigital syndromes (OPD1, OPD2), frontometaphyseal dysplasia (FMD), Melnick-Needles syndrome (MNS), and X-linked congenital idiopathic intestinal pseudoobstruction (CIIPX). Two transcript variants encoding different isoforms have been found for this gene.[provided by RefSeq, Mar 2009]
UniProt Code:	P21333
NCBI GenInfo Identifier:	116241365
NCBI Gene ID:	2316
NCBI Accession:	P21333.4
UniProt Secondary Accession:	P21333,Q5HY53, Q5HY55, Q8NF52, E9KL45,
UniProt Related Accession:	P21333
Molecular Weight:	280,739 Da
NCBI Full Name:	Filamin-A
NCBI Synonym Full Names:	filamin A, alpha
NCBI Official Symbol:	FLNA
NCBI Official Synonym Symbols:	FLN; FMD; MNS; OPD; ABPX; CSBS; CVD1; FLN1; NHBP; OPD1; OPD2; XLVD; XMVD; FLN-A; ABP-280
NCBI Protein Information:	filamin-A; filamin-1; alpha-filamin; non-muscle filamin; actin binding protein 280; endothelial actin-binding protein
UniProt Protein Name:	Filamin-A
UniProt Synonym Protein Names:	Actin-binding protein 280; ABP-280; Alpha-filamin; Endothelial actin-binding protein; Filamin-1; Non-muscle filamin
Protein Family:	Filamin
UniProt Gene Name:	FLNA
UniProt Entry Name:	FLNA_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-Filamin A 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.)