hnRNP Q Colorimetric Cell-Based ELISA (CBCAB00941)

Product Name:	hnRNP Q Colorimetric Cell-Based ELISA
Product Code:	CBCAB00941
ELISA Type:	Cell-Based
Target:	hnRNP Q
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of hnRNP Q concentration is achieved by an indirect ELISA format. In essence, hnRNP Q is captured by hnRNP Q-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: 10492, UniProt ID: O60506, OMIM: 0, Unigene: Hs.472056/Hs.571177
Gene Symbol:	SYNCRIP
Sub Type:	None

UniProt Protein Function:	NSAP1: a heterogeneous nuclear ribonucleoprotein (hnRNP) implicated in mRNA processing mechanisms. A component of the spliceosome. Five alternatively spliced isoforms have been described. Isoform 1, 2 and 3 are associated with pre-mRNA, splicing intermediates and mature mRNA protein complexes. Isoform 1 binds to apoB mRNA AU-rich sequences. Isoform 1 is part of the APOB mRNA editosome complex and may modulate the postranscriptional C to U RNA-editing of the APOB mRNA through either by binding to ACF (APOBEC1 complementation factor), to APOBEC1 or to RNA itself. May be involved in translationally coupled mRNA turnover. Implicated with other RNA-binding proteins in the cytoplasmic deadenylation/translational and decay interplay of the FOS mRNA mediated by the major coding-region determinant of instability (mCRD) domain. Interacts in vitro preferentially with poly(A) and poly(U) RNA sequences. Isoform 3 may be involved in cytoplasmic vesicle-based mRNA transport through interaction with synaptotagmins.
UniProt Protein Details:	Protein type:RNA splicing; Motility/polarity/chemotaxis; Spliceosome; RNA-binding Chromosomal Location of Human Ortholog: 6q14-q15 Cellular Component: membrane; nucleus; ribonucleoprotein complex Molecular Function:protein binding; RNA binding Biological Process: negative regulation of translation; nuclear mRNA splicing, via spliceosome; osteoblast differentiation; RNA processing; RNA splicing
NCBI Summary:	This gene encodes a member of the cellular heterogeneous nuclear ribonucleoprotein (hnRNP) family. hnRNPs are RNA binding proteins that complex with heterogeneous nuclear RNA (hnRNA) and regulate alternative splicing, polyadenylation, and other aspects of mRNA metabolism and transport. The encoded protein plays a role in multiple aspects of mRNA maturation and is associated with several multiprotein complexes including the apoB RNA editing-complex and survival of motor neurons (SMN) complex. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene, and a pseudogene of this gene is located on the short arm of chromosome 20. [provided by RefSeq, Dec 2011]
UniProt Code:	O60506
NCBI GenInfo Identifier:	92090361
NCBI Gene ID:	10492
NCBI Accession:	O60506.2
UniProt Secondary Accession:	O60506,Q53H05, Q5TCG2, Q5TCG3, Q8IW78, Q8N599, Q96LC1 Q96LC2, Q9Y583, E1P501, E1P502,
UniProt Related Accession:	O60506
Molecular Weight:	46,328 Da
NCBI Full Name:	Heterogeneous nuclear ribonucleoprotein Q
NCBI Synonym Full Names:	synaptotagmin binding cytoplasmic RNA interacting protein
NCBI Official Symbol:	SYNCRIP
NCBI Official Synonym Symbols:	PP68; NSAP1; GRYRBP; HNRNPQ; HNRPQ1; GRY-RBP; hnRNP-Q
NCBI Protein Information:	heterogeneous nuclear ribonucleoprotein Q
UniProt Protein Name:	Heterogeneous nuclear ribonucleoprotein Q
UniProt Synonym Protein Names:	Glycine- and tyrosine-rich RNA-binding protein; GRY-RBP; NS1-associated protein 1; Synaptotagmin-binding, cytoplasmic RNA-interacting protein
Protein Family:	Heterogeneous nuclear ribonucleoprotein
UniProt Gene Name:	SYNCRIP
UniProt Entry Name:	HNRPQ_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-hnRNP Q 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.)