Rat Neurofilament light polypeptide/NEFL ELISA Kit

Product Name:	Rat NEFL (Neurofilament, Light Polypeptide) ELISA Kit
Product Code:	RTFI00986
Size:	96 Assays
Target:	Rat NEFL
Alias:	NEFL, NF-L, 68 kDa neurofilament protein, Neurofilament triplet L protein
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat NEFL and the recovery rates were calculated by comparing the measured value to the expected amount of Rat NEFL in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-105	93
EDTA plasma(n=5)	86-104	94
UFH plasma(n=5)	92-103	99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat NEFL and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-104%	92-100%	88-101%
EDTA plasma(n=5)	82-100%	83-98%	82-92%
UFH plasma(n=5)	81-91%	84-100%	80-99%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P19527

UniProt Protein Function:	NFL: one of the three (L, M, and H) intermediate filament proteins that form neurofilaments. Neurofilaments are involved in the maintenance of neuronal caliber. NF-L is the most abundant of the three neurofilament proteins. Defects cause Charcot-Marie-Tooth disease type 1F (CMT1F).
UniProt Protein Details:	Protein type:Cytoskeletal Cellular Component: TSC1-TSC2 complex; neuron projection; growth cone; axon; cytoplasm; neurofilament; intermediate filament; myelin sheath Molecular Function:protein binding, bridging; protein C-terminus binding; protein domain specific binding; identical protein binding; phospholipase binding; structural constituent of cytoskeleton Biological Process: protein polymerization; response to peptide hormone stimulus; neurofilament bundle assembly; response to toxin; intermediate filament organization; intermediate filament bundle assembly; microtubule cytoskeleton organization and biogenesis; response to organic nitrogen; retrograde axon cargo transport; axon regeneration in the peripheral nervous system; response to organic substance; intermediate filament polymerization and/or depolymerization; axon transport of mitochondrion; neurofilament cytoskeleton organization and biogenesis; spinal cord development; regulation of axon diameter; positive regulation of axonogenesis; negative regulation of neuron apoptosis; locomotion; neuromuscular process controlling balance; anterograde axon cargo transport; neurite morphogenesis; response to corticosterone stimulus
NCBI Summary:	one component of the hamartin-tuberin tuberous sclerosis complex; may play a role in in central nervous system function [RGD, Feb 2006]
UniProt Code:	P19527
NCBI GenInfo Identifier:	13929098
NCBI Gene ID:	83613
NCBI Accession:	NP_113971.1
UniProt Secondary Accession:	P19527,Q63367,
UniProt Related Accession:	P19527
Molecular Weight:	61,335 Da
NCBI Full Name:	neurofilament light polypeptide
NCBI Synonym Full Names:	neurofilament, light polypeptide
NCBI Official Symbol:	Nefl
NCBI Official Synonym Symbols:	Nfl; NF-L
NCBI Protein Information:	neurofilament light polypeptide; 68 kDa neurofilament protein; neurofilament triplet L protein
UniProt Protein Name:	Neurofilament light polypeptide
UniProt Synonym Protein Names:	68 kDa neurofilament protein; Neurofilament triplet L protein
Protein Family:	Neurofilament light polypeptide
UniProt Gene Name:	Nefl
UniProt Entry Name:	NFL_RAT

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37°C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum:	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum and assay promptly or aliquot and store the samples at-80°C. Avoid multiple freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anti-coagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid:	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell Culture Supernatant:	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell Lysates:	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue Homogenates:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenizein 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or-80°C.
Tissue Lysates:	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk:	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.