Rat Csnk2a1 ELISA Kit (RTEB0559)- High Sensitivity

Product Name:	Rat Casein kinase II subunit alpha (Csnk2a1) ELISA Kit
SKU:	RTEB0559
Size:	96T
Target:	Rat Casein kinase II subunit alpha (Csnk2a1)
Synonyms:	CK II alpha
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	78-5000pg/mL
Sensitivity:	34pg/mL

Intra CV:

3.8%

Inter CV:

7.5%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	95-105%	102-111%	101-110%	94-104%
EDTA Plasma(N=5)	100-110%	103-113%	96-104%	99-108%
Heparin Plasma(N=5)	99-107%	108-118%	101-112%	95-105%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	80	80-85
Plasma	81	80-87

Function:

Catalytic subunit of a constitutively active serine/threonine-protein kinase complex that phosphorylates a large number of substrates containing acidic residues C-terminal to the phosphorylated serine or threonine. Regulates numerous cellular processes, such as cell cycle progression, apoptosis and transcription, as well as viral infection. May act as a regulatory node which integrates and coordinates numerous signals leading to an appropriate cellular response. During mitosis, functions as a component of the p53/TP53-dependent spindle assembly checkpoint (SAC) that maintains cyclin-B-CDK1 activity and G2 arrest in response to spindle damage. Also required for p53/TP53-mediated apoptosis, phosphorylating 'Ser-392' of p53/TP53 following UV irradiation. Can also negatively regulate apoptosis. Phosphorylates the caspases CASP9 and CASP2 and the apoptotic regulator NOL3. Phosphorylation protects CASP9 from cleavage and activation by CASP8, and inhibits the dimerization of CASP2 and activation of CASP8. Regulates transcription by direct phosphorylation of RNA polymerases I, II, III and IV. Also phosphorylates and regulates numerous transcription factors including NF-kappa-B, STAT1, CREB1, IRF1, IRF2, ATF1, SRF, MAX, JUN, FOS, MYC and MYB. Phosphorylates Hsp90 and its co-chaperones FKBP4 and CDC37, which is essential for chaperone function. Regulates Wnt signaling by phosphorylating CTNNB1 and the transcription factor LEF1. Acts as an ectokinase that phosphorylates several extracellular proteins. Phosphorylates PML at 'Ser-565' and primes it for ubiquitin-mediated degradation (By similarity). Plays an important role in the circadian clock function by phosphorylating ARNTL/BMAL1 at 'Ser-90' which is pivotal for its interaction with CLOCK and which controls CLOCK nuclear entry. Phosphorylates CCAR2 at 'Thr-454'.

Uniprot:	P19139
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Casein kinase II subunit alpha
Sub Unit:	Heterotetramer composed of two catalytic subunits (alpha chain and/or alpha' chain) and two regulatory subunits (beta chains). The tetramer can exist as a combination of 2 alpha/2 beta, 2 alpha'/2 beta or 1 alpha/1 alpha'/2 beta subunits. Also part of a CK2-SPT16-SSRP1 complex composed of SSRP1, SUPT16H, CSNK2A1, CSNK2A2 and CSNK2B, which forms following UV irradiation. Interacts with RNPS1, SNAI1, PML and CCAR2.
Research Area:	Cancer
Subcellular Location:	Nucleus
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	CK2A1: an ubiquitous protein kinase of the CK2 family. Exists as a tetramer composed of two catalytic subunits, alpha and alpha-prime, and two regulatory beta subunits. The beta subunits undergo autophosphorylation. The isoforms are rarely specified in publications. Two splice variant isoforms have been found. Participates in Wnt signaling. Phosphorylates E2 ubiquitin conjugating enzyme UBC3B inducing its interaction with beta-TRCp and enhancing beta-catenin degradation. May control IkB-alpha and p27Kip1 degradation. component of a CK2-SPT16-SSRP1 complex composed of SSRP1, PT16 CK2-A1, CK2-A2 and CK2-B the complex associating following UV irradiation. Interacts with RNPS1. Mouse transgene causes mammary gland hyperplasia and lymphoma, and activation by bovine parasites leads to fatal lymphoproliferation. Expression and activity are elevated in lung tumors and breast tumors. Antisense drives apoptosis of tumor cell lines and xenografts. Involved in DNA break repair by phosphorylation of scaffold protein XRCC1, phosphorylation of BRCA1, and phosphorylation of p53 in response to UV irradiation. Drosophila CK2 (Timekeeper) is involved in circadian regulation. Phosphorylates and binds to a major component of the inclusion bodies seen in Parkinson?s patients. Inhibitors: 4,5,6,7-tetrabromobenzotriazole (TBB).
UniProt Protein Details:	Protein type:Protein kinase, Ser/Thr (non-receptor); Kinase, protein; EC 2.7.11.1; Protein kinase, Other; Other group; CK2 family Cellular Component: nucleus; NuRD complex; PcG protein complex; protein kinase CK2 complex; Sin3 complex Molecular Function:beta-catenin binding; kinase activity; protein N-terminus binding; protein phosphatase regulator activity; protein serine/threonine kinase activity Biological Process: negative regulation of caspase activity; positive regulation of cell growth; positive regulation of cell proliferation; positive regulation of protein catabolic process; positive regulation of Wnt receptor signaling pathway; protein amino acid autophosphorylation; protein amino acid phosphorylation
NCBI Summary:	Casein kinase II is a serine/threonine protein kinase that phosphorylates acidic proteins such as casein. It is involved in various cellular processes, including cell cycle control, apoptosis, and circadian rhythms. The kinase exists as a tetramer and is composed of an alpha, an alpha-prime, and two beta subunits. The alpha subunits contain the catalytic activity while the beta subunits undergo autophosphorylation. The protein encoded by this gene represents the alpha subunit. [provided by RefSeq, Feb 2014]
UniProt Code:	P19139
NCBI GenInfo Identifier:	729878
NCBI Gene ID:	116549
NCBI Accession:	P19139.2
UniProt Secondary Accession:	P19139,Q5BKC1,
UniProt Related Accession:	P19139
Molecular Weight:	45,073 Da
NCBI Full Name:	Casein kinase II subunit alpha
NCBI Synonym Full Names:	casein kinase 2 alpha 1
NCBI Official Symbol:	Csnk2a1
NCBI Protein Information:	casein kinase II subunit alpha
UniProt Protein Name:	Casein kinase II subunit alpha
Protein Family:	Casein kinase
UniProt Gene Name:	Csnk2a1
UniProt Entry Name:	CSK21_RAT

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.