PKC zeta (Phospho-Thr560)Colorimetric Cell-Based ELISA Kit (CBCAB01378)

Product Name:	PKC zeta (Phospho-Thr560)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01378
ELISA Type:	Cell-Based
Target:	PKC zeta (Phospho-Thr560)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The PKC zeta (Phospho-Thr560) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PKC zeta protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PKC zeta in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PKC zeta phosphorylation.

Qualitative determination of PKC zeta (Phospho-Thr560) concentration is achieved by an indirect ELISA format. In essence, PKC zeta (Phospho-Thr560) is captured by PKC zeta (Phospho-Thr560)-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: 5590, UniProt ID: Q05513, OMIM: 176982, Unigene: Hs.496255
Gene Symbol:	PRKCZ
Sub Type:	Phospho

UniProt Protein Function:	PKCZ: an AGC kinase of the PKC family. An atypical PKC: its activity is not regulated by Ca2+, PS, DAG or phorbol esters. Constitutively active.
UniProt Protein Details:	Protein type:Kinase, protein; Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; EC 2.7.11.13; AGC group; PKC family; Iota subfamily Chromosomal Location of Human Ortholog: 1p36.33-p36.2 Cellular Component: cell junction; cytoplasm; cytosol; intercellular junction; membrane; plasma membrane; vesicle Molecular Function:insulin receptor substrate binding; protein binding; protein kinase activity; protein kinase C activity; protein serine/threonine kinase activity Biological Process: activation of NF-kappaB transcription factor; establishment of cell polarity; negative regulation of insulin receptor signaling pathway; negative regulation of peptidyl-tyrosine phosphorylation; negative regulation of protein complex assembly; peptidyl-serine phosphorylation; positive regulation of insulin receptor signaling pathway; positive regulation of interleukin-4 production; positive regulation of T-helper 2 cell differentiation; protein amino acid phosphorylation; signal transduction; transforming growth factor beta receptor signaling pathway
NCBI Summary:	Protein kinase C (PKC) zeta is a member of the PKC family of serine/threonine kinases which are involved in a variety of cellular processes such as proliferation, differentiation and secretion. Unlike the classical PKC isoenzymes which are calcium-dependent, PKC zeta exhibits a kinase activity which is independent of calcium and diacylglycerol but not of phosphatidylserine. Furthermore, it is insensitive to typical PKC inhibitors and cannot be activated by phorbol ester. Unlike the classical PKC isoenzymes, it has only a single zinc finger module. These structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from other isoenzymes of PKC. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]
UniProt Code:	Q05513
NCBI GenInfo Identifier:	68067736
NCBI Gene ID:	5590
NCBI Accession:	Q05513.4
UniProt Secondary Accession:	Q05513,Q15207, Q5SYT5, Q969S4, A8K4N0, A8MU64, B7Z2J7 E9PCW2,
UniProt Related Accession:	Q05513
Molecular Weight:	56,136 Da
NCBI Full Name:	Protein kinase C zeta type
NCBI Synonym Full Names:	protein kinase C zeta
NCBI Official Symbol:	PRKCZ
NCBI Official Synonym Symbols:	PKC2; PKC-ZETA
NCBI Protein Information:	protein kinase C zeta type
UniProt Protein Name:	Protein kinase C zeta type
UniProt Synonym Protein Names:	nPKC-zeta
Protein Family:	Protein kinase
UniProt Gene Name:	PRKCZ
UniProt Entry Name:	KPCZ_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-PKC zeta (Phospho-Thr560) Antibody, Anti-PKC zeta 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.)