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HTRF Human Total Nucleolin Detection Kit, 500 assay points

Total Nucleolin Product image
HTRF Human Total Nucleolin Detection Kit, 500 assay points
Total Nucleolin Product image
Total Nucleolin Product image
Total Nucleolin Product image
Total Nucleolin Product image
Total Nucleolin Product image

This HTRF kit allows for the cell-based quantitative detection of total Nucleolin.

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MARCH10
Terms and conditions apply.
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Feature Specification
Application Cell Signaling
Sample Volume 16 µL
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This HTRF kit allows for the cell-based quantitative detection of total Nucleolin.

Click to copy promo code to clipboard.
March lab savings - SAVE 10%. Use promo code below.
MARCH10
Terms and conditions apply.
View product information
Product Variants
Unit Size: 500 assay points
Part #:
64NCLTPEG
List Price
USD 2,147.00
Your online price:
Unit Size: 10,000 assay points
Part #:
64NCLTPEH
List Price
USD 12,490.00
Your online price:
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
Total list price:
USD 2,147.00
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Total Nucleolin Product image
Total Nucleolin Product image
HTRF Human Total Nucleolin Detection Kit, 500 assay points
Total Nucleolin Product image
Total Nucleolin Product image
Total Nucleolin Product image
HTRF Human Total Nucleolin Detection Kit, 500 assay points
Total Nucleolin Product image
Total Nucleolin Product image

Product information

Overview

Nucleolin is protein that is mostly localized in the nucleus, where it is involved in ribosome biogenesis by binding to and stabilizing pre-RNA. It also plays several other roles in conjunction with ribosomes, such as RNA transcription regulation via transcription factor binding and nucleus-cytosol mRNA trafficking. The phosphorylation state of nucleolin regulates its activity by increasing its binding affinity for RNA, and is especially enhanced in response to cellular stress and DNA damage. Abnormal phosphorylation of nucleolin is recognized as a potential source of lack of regulation of mRNA transcription, which can alter the expression of pro- and anti-oncogenic genes and contribute to cancer development or progression.

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
HTRF
Detection Modality
HTRF
Lysis Buffer Compatibility
Lysis Buffer 2
Molecular Modification
Total
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target
Nucleolin
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
TR-FRET
Therapeutic Area
Inflammation
Oncology
Unit Size
500 assay points

How it works

Total Nucleolin assay principle

The Total Nucleolin assay measures Nucleolin levels in cells. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. In the presence of Nucleolin, the labeled antibodies form an immune complex, bringing the donor fluorophore into close proximity to the acceptor and generating a FRET signal. The signal intensity is directly proportional to the concentration of total protein present in the sample.

generic-R-D-attributes-assay-principle-total-with-phospho.png
 
Total Nucleolin two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total Nucleolin HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

generic-R-D-attributes-assay-protocol-how-it-works-2-plates.png
 
Total Nucleolin one-plate assay protocol

Detection of Total Nucleolin with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol allows miniaturization while maintaining robust HTRF quality.

generic-R-D-attributes-assay-protocol-how-it-works-1-plate.png

Assay validation

Induction of Nucleolin phosphorylation with Calyculin in HeLa cells

Hela cells were plated at 40,000 cells/well in 100 µL in a 96-well plate in plain Eagle’s Minimum Essential medium, and incubated 24 hours at 37°C, 5% CO2.

The cells were stimulated with varying doses of Calyculin A for 1 hour, and then lysed with 100ul of 1x lysis buffer #2 for 30 minutes at RT under gentle shaking.

Once the lysis was complete, 16 µL of lysate were transferred into a low volume white microplate before the addition of 2 µL each of the donor and acceptor HTRF Nucleolin detection reagents. The HTRF signal was recorded after overnight incubation.

assay-validation-activator-calyculin-Nucleolin-Phospho-T84-64NCLT84PEG-64NCLT84PEH-64NCLT84PEY.jpg

Simplified pathway

Nucleolin signaling pathway

Nucleolin is heavily involved in the regulation of the cell cycle due to its close collaboration with the ribosomal units that perform mRNA translation into proteins. In normal circumstances, nucleolin can be involved in the regulation of every step of the DNA transcription to mRNA translation process. It binds transcription complexes directly, then picks up the resulting RNA and promotes its folding and assembly into ribosomes for pre-RNA ribosomal units or its translocation to the cytosol for mRNA. For the latter, it increases the stability in the cytosol and supports the interaction between mRNA and ribosomes. As such, nucleolin is a critical regulator of gene expression and is itself under regulation depending on its state of phosphorylation. Several phosphorylation sites attune its properties toward a given role in the transcription-translation chain and have been associated with some cancers when the phosphorylation patterns of nucleolin are abnormal.

Simplified-pathway-Nucleolin-Total-64NCLT84PEG-64NCLT84PEH-64NCLT84PEY.jpg

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