Human NF-L DuoSet ELISA

R&D Systems | Catalog # DY1644-05

Human NF-L DuoSet ELISA, 5 Plate
R&D Systems
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Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

125-8000 pg/mL

Sample Type

Cell culture supernates, serum, and plasma
Note: Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet

Reactivity

Human

Human NF-L DuoSet ELISA Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits
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Product Summary for Human NF-L DuoSet ELISA

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human NF-L. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma should be evaluated prior to use in this DuoSet.

Product Specifications

Assay Format

96-well strip plate (sold separately)

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Unconjugated

Label

HRP

Scientific Data Images for Human NF-L DuoSet ELISA

Human NF-L DuoSet Standard Curve

Human NF-L DuoSet Standard Curve

Kit Contents for Human NF-L DuoSet ELISA

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2(5 plates):(Catalog #DY008C) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

The components listed above may be purchased separately:

PBS:(Catalog #DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer:(Catalog #WA126), or equivalent

Reagent Diluent*

Blocking Buffer*

Substrate Diluent:ELISA TMB Substrate R&DSystems, (Catalog#DY999B or DY999B-250)

ELISA Stop Solution:Methanesulfonic acid R&D Systems, (Catalog # DY994B or DY994B-250)

Microplates: R&D Systems (Catalog #DY990)

Plate Sealers:ELISA Plate Sealers (Catalog #DY992)

*For the Reagent Diluent and Blocking Buffer arerecommended for a specific DuoSet ELISA Development Kit,see the product datasheet.

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Background: NF-L

Neurofilaments are intermediate filaments that are major components of the neuronal cytoskeleton. Neurofilaments are composed of three subunits: the heavy subunit (NF-H), medium subunit (NF-M), and light subunit (NF-L). Neurofilaments are trimers that always contain the 68 kDa NF-L and variably contain 125 kDa NF-M and 200 kDa NF-H. The human neurofilament heavy chain is a phosphorylated cytoskeletal intermediate filament protein that is expressed in neurons. The neurofilament medium subunit Human NF-M is a phosphorylated glycoprotein that belongs to the type IV intermediate filament family. NF-L is a 68 kDa light chain cytoskeletal intermediate filament protein that associates with the 125 kDa NF-M and the 200 kDa NF-H to form neurofilaments.

All three neurofilament subunits share a common structure, with globular head, alpha-helical rod, and carboxy terminal side-arm domains. The subunits assemble to form 10 nm neurofilaments. Accumulation of neurofilaments is seen in several human diseases including amyotrophic lateral sclerosis (ALS), Parkinsons disease, Alzheimers disease, and diabetic neuropathy.

Long Name

Neurofilament Protein, Light Chain

Alternate Names

CMT2E, NEFL, NF68, NFL

Entrez Gene IDs

4747 (Human)

Gene Symbol

NEFL

Additional NF-L Products

Product Documents for Human NF-L DuoSet ELISA

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Human NF-L DuoSet ELISA

For research use only

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Protocols

View specific protocols for Human NF-L DuoSet ELISA (DY1644-05):

GENERAL ELISA PROTOCOL

Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 100 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

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