Figure 1. Detection of Neurofilament-L protein in mouse brain, HeLa cells, and rat brain is examined by using the PathScan® Total Neurofilament Sandwich ELISA Kit. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blot using Neurofilament-L (C28E10) Rabbit mAb #2837 is shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from mouse brain and the absorbance at 450 nm as detected by the PathScan® Total Neurofilament-L Sandwich ELISA Kit is shown. Mouse brain was harvested and then lysed with the lysis buffer #9803.
|Product Includes||Volume||Solution Color|
|Neurofilament-L Mouse mAb Coated Microwells||96 tests|
|Neurofilament-L Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total Neurofilament-L Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of neurofilament-L protein. A neurofilament-L mouse mAb has been coated onto the microwells. After incubation with cell lysates, the neurofilament-L proteins are captured by the coated antibody. Following extensive washing, an neurofilament-L rabbit detection mAb is added to detect captured neurofilament-L proteins. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for the developed color is proportional to the quantity of neurofilament-L protein.
Antibodies in the kit are custom formulations specific to the kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total Neurofilament-L Sandwich ELISA Kit detects endogenous levels of nerofilament-L protein in mouse or rat brains, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
The cytoskeleton consists of three types of cytosolic fibers: actin microfilaments, intermediate filaments, and microtubules. Neurofilaments are the major intermediate filaments found in neurons and consist of light (NFL), medium (NFM), and heavy (NFH) subunits (1). Similar in structure to other intermediate filament proteins, neurofilaments have a globular amino-terminal head, a central α-helical rod domain, and a carboxy-terminal tail. A heterotetrameric unit (NFL-NFM and NFL-NFH) forms a protofilament, with eight protofilaments comprising the typical 10 nm intermediate filament (2). While neurofilaments are critical for radial axon growth and determine axon caliber, microtubules are involved in axon elongation. PKA phosphorylates the head domain of NFL and NFM to inhibit neurofilament assembly (3,4). Research studies have shown neurofilament accumulations in many human neurological disorders including Parkinson's disease (in Lewy bodies along with α-synuclein), Alzheimer's disease, Charcot-Marie-Tooth disease, and Amyotrophic Lateral Sclerosis (ALS) (1).
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