FluoBolt-Noggin Fluorescence Immunoassay
The FluoBolt Noggin Fluorescence Immunoassay is manufactured in Austria by Fianostics GmbH
Method: Metal Enhanced Direct Sandwich Fluorescence Immunoassay
Size: 1×96 wells
Sensitivity: LOD <0 pmol/l + 3 SD): 10 pmol/l; LLOQ: 5 pmol/l
Dynamic Range: 0-250 pmol/l
Incubation Time: Overnight
Sample Type: Serum, Plasma
Sample Size: 20 µl
For Research Use Only
This platform is fully compatible to standard laboratory methodology using 96 well microtiter plate format and assays based on this technology can be run on any standard fluorescence microplate reader.
This FluoBolt-Noggin Fluorescence Immunoassay is regularly delivered standard with AlexaFluor680 labeled detection-antibody. If you wish to use a different dye, the following options are available
- FITC labeled (FIA-1701-F)
- Cy3 labeled (FIA-1701-C3)
- Cy5 labeled (FIA-1701-C5)
Conversion factor: 1 pg/ml = 0.015 pmol/l (MW: 66 kD)
Noggin is a secreted homodimeric glycoprotein that is an antagonist of bone morphogenetic proteins (BMPs). Human Noggin cDNA encodes a 232 amino acid (aa) precursor protein; cleavage of a 19 aa signal peptide generates the 213 aa mature protein which contains an N-terminal acidic region, a central basic heparin-binding segment and a C-terminal cysteine-knot structure. Secreted Noggin probably remains close to the cell surface due to its binding of heparin containing proteoglycans. Noggin is very highly conserved among vertebrates.
Noggin predominantly binds BMP-4 and BMP-2 and antagonizes their bioactivities by preventing binding to both type I and type II receptors. During embryogenesis Noggin is a crucial factor for regulation of various developmental processes like formation of neural tubes, cardiomyocyte growth and patterning as well as skeletal development where Noggin prevents chondrocyte hyperplasia, thus allowing proper formation of joints. Mutations within the cysteine-knot region of human Noggin are linked to multiple types of skeletal dysplasias that result in apical joint fusions. Noggin is expressed in defined areas of the adult central nervous system and peripheral tissues such as lung, skeletal muscle and skin.
NOGGIN has been linked to:
- Bone Tumors
- Ankylosing Spondylitis
- Non Alcoholic Fatty Liver Disease (NAFLD)
- Pulmonary Arterial Hypertension (PAH)
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About Metal Enhanced Fluorescence
Metal Enhanced Fluorescence (MEF) using metal nano-structures offers the possibility to increase the analytical sensitivity of systems based on Iuorescence detection dramatically. FIANOSTICS has developed a new MEF- immunoassay platform, that allows up to 300 fold gains of sensitivity. This platform is fully compatible to standard laboratory methodology using 96 well microtiter plate format and assays based on this technology can be run on any standard fIuorescence microplate reader. Its unique features enable us to develop high sensitivity, single step with no wash florescence immunoassays for low abundance biomarkers.
All reagents must be at room temperature (18-26C) before use in this assay
- Add 25 μl of the selected fluorescence labeled detection antibody (DAF or DA3 or DA5 or DAA) to all wells required. Swirl gently.
- Add 20 μl of standard, control or sample to the wells according to the marked positions on the protocol sheet, swirl gently, cover tightly with the delivered adhesive film and incubate for 4 hours at 37°C, or over night at room temperature (18-26°C) in the dark.
- a)If your reader allows bottom reading, read the plate without any further processing at the Ex/Em wavelength fitting to the delivered detection antibody (495/518 nm for DAF, 550/570 nm for DA3, 650/670 nm for DA5, 679/702 nm for DAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signal of the 0 pM and the 250 pM NOGGIN standard. Samples with signals exceeding the signal of the highest standard must be re-run with an appropriate dilution using sample diluent (DD).
b)If your reader has no bottom read option or if you want to store the plate for documentation purposes, discard or aspirate the content of the wells and wash 3x with diluted wash buffer. Use a minimum of 200 µl wash buffer per well. After the final wash, remove remaining fluid by strongly tapping the plate against a paper towel. Read the plate in top configuration without any further processing at the Ex/Em wavelength fitting to the chosen detection antibody (495/518 nm for DAF, 550/570 nm for DA3, 650/670 nm for DA5, 679/702 nm for DAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signals of the 0 pM and the 250 pM NOGGIN standard. Samples with signals exceeding the signal of the highest standard must be re-run with appropriate dilution using sample diluent (DD).
Quality of bottom reading (3a) may vary between microplate readers. For first time users we suggest to perform the washing step and follow protocol 3b (ED).
- Store the plate with desiccant at 4°C (2-8°C) in the aluminium bag. Unused wells are stable until expiry date stated on the label. Fluorescence signals of standards, controls and samples remain detectable for at least two month at the plate surface, depending on signal intensity achieved.