AKAP5 Colorimetric Cell-Based ELISA (CBCAB00992)

Product Name:	AKAP5 Colorimetric Cell-Based ELISA
Product Code:	CBCAB00992
ELISA Type:	Cell-Based
Target:	AKAP5
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The AKAP5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect AKAP5 protein expression profile in cells. The kit can be used for measuring the relative amounts of AKAP5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on AKAP5.

Qualitative determination of AKAP5 concentration is achieved by an indirect ELISA format. In essence, AKAP5 is captured by AKAP5-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: 9495, UniProt ID: P24588, OMIM: 604688, Unigene: Hs.656683
Gene Symbol:	AKAP5
Sub Type:	None

UniProt Protein Function:	AKAP5: May anchor the PKA protein to cytoskeletal and/or organelle-associated proteins, targeting the signal carried by cAMP to specific intracellular effectors. Association with to the beta2-adrenergic receptor (beta2-AR) not only regulates beta2-AR signaling pathway, but also the activation by PKA by switching off the beta2-AR signaling cascade. Binding protein for dimer of the RII-beta regulatory subunit of cAMP-dependent protein kinase (PKA) and also for the protein kinase C (PKC) and the phosphatase calcineurin (PP2B). Each enzyme is inhibited when bound to the anchoring protein. Also binds the beta2-adrenergic receptor. Part of a complex containing AKAP5, ADCY5, ADCY6 AND PDE4C. Interacts with ADCY8, and enhances its phosphorylation at lipid rafts. Predominantly in the cerebral cortex and the postsynaptic densities of the forebrain, and to a lesser extent in adrenal medulla, lung and anterior pituitary.
UniProt Protein Details:	Protein type:Adaptor/scaffold Chromosomal Location of Human Ortholog: 14q23.3 Cellular Component: cytosol; plasma membrane Molecular Function:adenylate cyclase binding; protein binding; protein kinase A binding Biological Process: positive regulation of cAMP biosynthetic process; synaptic transmission
NCBI Summary:	The A-kinase anchor proteins (AKAPs) are a group of structurally diverse proteins, which have the common function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. This gene encodes a member of the AKAP family. The encoded protein binds to the RII-beta regulatory subunit of PKA, and also to protein kinase C and the phosphatase calcineurin. It is predominantly expressed in cerebral cortex and may anchor the PKA protein at postsynaptic densities (PSD) and be involved in the regulation of postsynaptic events. It is also expressed in T lymphocytes and may function to inhibit interleukin-2 transcription by disrupting calcineurin-dependent dephosphorylation of NFAT. [provided by RefSeq, Jul 2008]
UniProt Code:	P24588
NCBI GenInfo Identifier:	281185503
NCBI Gene ID:	9495
NCBI Accession:	P24588.3
UniProt Secondary Accession:	P24588,A2RRB8,
UniProt Related Accession:	P24588
Molecular Weight:	47,088 Da
NCBI Full Name:	A-kinase anchor protein 5
NCBI Synonym Full Names:	A-kinase anchoring protein 5
NCBI Official Symbol:	AKAP5
NCBI Official Synonym Symbols:	H21; AKAP75; AKAP79
NCBI Protein Information:	A-kinase anchor protein 5
UniProt Protein Name:	A-kinase anchor protein 5
UniProt Synonym Protein Names:	A-kinase anchor protein 79 kDa; AKAP 79; H21; cAMP-dependent protein kinase regulatory subunit II high affinity-binding protein
Protein Family:	A-kinase anchor protein
UniProt Gene Name:	AKAP5
UniProt Entry Name:	AKAP5_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-AKAP5 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.)