NBL1 Colorimetric Cell-Based ELISA (CBCAB01012)

Product Name:	NBL1 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01012
ELISA Type:	Cell-Based
Target:	NBL1
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The NBL1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NBL1 protein expression profile in cells. The kit can be used for measuring the relative amounts of NBL1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NBL1.

Qualitative determination of NBL1 concentration is achieved by an indirect ELISA format. In essence, NBL1 is captured by NBL1-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: 4681, UniProt ID: P41271, OMIM: 600613, Unigene: Hs.654502
Gene Symbol:	NBL1
Sub Type:	None

UniProt Protein Function:	NBL1 iso2: Possible candidate as a tumor suppressor gene of neuroblastoma. May play an important role in preventing cells from entering the final stage (G1/S) of the transformation process. Belongs to the DAN family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Tumor suppressor; Secreted Chromosomal Location of Human Ortholog: 1p36.13 Cellular Component: extracellular space Molecular Function:morphogen activity; protein homodimerization activity Biological Process: nervous system development; determination of dorsal identity; positive regulation of neuron differentiation; neurite morphogenesis; negative regulation of BMP signaling pathway
NCBI Summary:	This gene product is the founding member of the evolutionarily conserved CAN (Cerberus and DAN) family of proteins, which contain a domain resembling the CTCK (C-terminal cystine knot-like) motif found in a number of signaling molecules. These proteins are secreted, and act as BMP (bone morphogenetic protein) antagonists by binding to BMPs and preventing them from interacting with their receptors. They may thus play an important role during growth and development. Alternatively spliced transcript variants have been identified for this gene. Read-through transcripts between this locus and the upstream mitochondrial inner membrane organizing system 1 gene (GeneID 440574) have been observed. [provided by RefSeq, May 2013]
UniProt Code:	P41271
NCBI GenInfo Identifier:	323276675
NCBI Gene ID:	4681
NCBI Accession:	NP_001191013.1
UniProt Secondary Accession:	P41271,Q5TGZ2, Q5U0N4, Q96L68, A3KFI7,
UniProt Related Accession:	P41271
Molecular Weight:	19,277 Da
NCBI Full Name:	neuroblastoma suppressor of tumorigenicity 1 isoform 2
NCBI Synonym Full Names:	neuroblastoma 1, DAN family BMP antagonist
NCBI Official Symbol:	NBL1
NCBI Official Synonym Symbols:	NB; DAN; NO3; DAND1; D1S1733E
NCBI Protein Information:	neuroblastoma suppressor of tumorigenicity 1; DAN domain family member 1; differential screening-selected gene aberrant in neuroblastoma; neuroblastoma candidate region, suppression of tumorigenicity 1
UniProt Protein Name:	Neuroblastoma suppressor of tumorigenicity 1
UniProt Synonym Protein Names:	DAN domain family member 1; Protein N03; Zinc finger protein DAN
Protein Family:	D-aminoacylase
UniProt Gene Name:	NBL1
UniProt Entry Name:	NBL1_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-NBL1 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.)