hnRNP K (Phospho-Ser284)Colorimetric Cell-Based ELISA Kit (CBCAB01450)

Product Name:	hnRNP K (Phospho-Ser284)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01450
ELISA Type:	Cell-Based
Target:	hnRNP K (Phospho-Ser284)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The hnRNP K (Phospho-Ser284) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect hnRNP K protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated hnRNP K in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on hnRNP K phosphorylation.

Qualitative determination of hnRNP K (Phospho-Ser284) concentration is achieved by an indirect ELISA format. In essence, hnRNP K (Phospho-Ser284) is captured by hnRNP K (Phospho-Ser284)-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: 3190, UniProt ID: P61978, OMIM: 600712, Unigene: Hs.522257
Gene Symbol:	HNRNPK
Sub Type:	Phospho

UniProt Protein Function:	hnRNP K: a heterogeneous nuclear ribonucleoprotein (hnRNP). One of the major pre-mRNA-binding proteins. Binds tenaciously to poly(C) sequences. Likely to play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences. Can also bind poly(C) single-stranded DNA. Two alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:RNA splicing; RNA-binding; Spliceosome Chromosomal Location of Human Ortholog: 9q21.32 Cellular Component: cell-cell adherens junction; extracellular matrix; focal adhesion; membrane; nuclear chromatin; nucleoplasm; nucleus Molecular Function:protein binding; RNA binding; single-stranded DNA binding Biological Process: gene expression; negative regulation of apoptosis; nuclear mRNA splicing, via spliceosome; positive regulation of low-density lipoprotein receptor biosynthetic process; positive regulation of receptor-mediated endocytosis; positive regulation of transcription from RNA polymerase II promoter; protein sumoylation; RNA processing; signal transduction Disease: Au-kline Syndrome
NCBI Summary:	This gene belongs to the subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are RNA binding proteins and they complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. While all of the hnRNPs are present in the nucleus, some seem to shuttle between the nucleus and the cytoplasm. The hnRNP proteins have distinct nucleic acid binding properties. The protein encoded by this gene is located in the nucleoplasm and has three repeats of KH domains that binds to RNAs. It is distinct among other hnRNP proteins in its binding preference; it binds tenaciously to poly(C). This protein is also thought to have a role during cell cycle progession. Several alternatively spliced transcript variants have been described for this gene, however, not all of them are fully characterized. [provided by RefSeq, Jul 2008]
UniProt Code:	P61978
NCBI GenInfo Identifier:	48429103
NCBI Gene ID:	3190
NCBI Accession:	P61978.1
UniProt Secondary Accession:	P61978,Q07244, Q15671, Q59F98, Q5T6W4, Q60577, Q922Y7 Q96J62,
UniProt Related Accession:	P61978
Molecular Weight:	48,562 Da
NCBI Full Name:	Heterogeneous nuclear ribonucleoprotein K
NCBI Synonym Full Names:	heterogeneous nuclear ribonucleoprotein K
NCBI Official Symbol:	HNRNPK
NCBI Official Synonym Symbols:	AUKS; CSBP; TUNP; HNRPK
NCBI Protein Information:	heterogeneous nuclear ribonucleoprotein K
UniProt Protein Name:	Heterogeneous nuclear ribonucleoprotein K
UniProt Synonym Protein Names:	Transformation up-regulated nuclear protein; TUNP
Protein Family:	Heterogeneous nuclear ribonucleoprotein
UniProt Gene Name:	HNRNPK

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-hnRNP K (Phospho-Ser284) Antibody, Anti-hnRNP K 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.)