Product Name:	Human pro-BDNF(pro Brain Derived Neurotrophic Factor) ELISA Kit
Product Code:	HUFI03121
Size:	96 Assays
Alias:	brain-derived neurotrophic factor isoform a preproprotein
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human pro-BDNF concentrations in serum plasma and other biological fluids.
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 Human pro-BDNF and the recovery rates were calculated by comparing the measured value to the expected amount of Human pro-BDNF in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human pro-BDNF and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
CV(%):	Intra-Assay: CV<8% Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P23560

UniProt Protein Function:	BDNF: During development, promotes the survival and differentiation of selected neuronal populations of the peripheral and central nervous systems. Participates in axonal growth, pathfinding and in the modulation of dendritic growth and morphology. Major regulator of synaptic transmission and plasticity at adult synapses in many regions of the CNS. The versatility of BDNF is emphasized by its contribution to a range of adaptive neuronal responses including long-term potentiation (LTP), long-term depression (LTD), certain forms of short-term synaptic plasticity, as well as homeostatic regulation of intrinsic neuronal excitability. Defects in BDNF are a cause of congenital central hypoventilation syndrome (CCHS); also known as congenital failure of autonomic control or Ondine curse. CCHS is a rare disorder characterized by abnormal control of respiration in the absence of neuromuscular or lung disease, or an identifiable brain stem lesion. A deficiency in autonomic control of respiration results in inadequate or negligible ventilatory and arousal responses to hypercapnia and hypoxemia. CCHS is frequently complicated with neurocristopathies such as Hirschsprung disease that occurs in about 16% of CCHS cases. Belongs to the NGF-beta family. 5 isoforms of the human protein are produced by alternative promoter.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Cell development/differentiation; Secreted; Cytokine Chromosomal Location of Human Ortholog: 11p13 Cellular Component: extracellular space; synaptic vesicle; mitochondrial crista; perinuclear region of cytoplasm; cytoplasmic membrane-bound vesicle; cytoplasm; dendrite; extracellular region; terminal button; perikaryon Molecular Function:growth factor activity; neurotrophin TRKB receptor binding Biological Process: circadian rhythm; axon guidance; mechanoreceptor differentiation; behavioral fear response; mitochondrial electron transport, NADH to ubiquinone; regulation of neuron differentiation; axon extension; response to hormone stimulus; positive regulation of long-term neuronal synaptic plasticity; response to vitamin A; negative regulation of neuroblast proliferation; ureteric bud development; dendrite development; regulation of retinal cell programmed cell death; feeding behavior; negative regulation of neuron apoptosis; response to electrical stimulus; negative regulation of synaptic transmission, GABAergic; inner ear development; response to food; nervous system development; chronic inflammatory response; response to light intensity; neuron recognition; regulation of short-term neuronal synaptic plasticity; learning; regulation of axon extension; negative regulation of striated muscle development; positive regulation of peptidyl-serine phosphorylation; positive regulation of synaptogenesis; axon target recognition; response to hyperoxia; glutamate secretion; response to hypoxia; response to fluoxetine; nerve development; response to activity; positive regulation of neuron differentiation; regulation of excitatory postsynaptic membrane potential; neurite morphogenesis; transmembrane receptor protein tyrosine kinase signaling pathway; gamma-aminobutyric acid signaling pathway Disease: Bulimia Nervosa, Susceptibility To, 1; Obsessive-compulsive Disorder; Bulimia Nervosa, Susceptibility To, 2; Central Hypoventilation Syndrome, Congenital
NCBI Summary:	The protein encoded by this gene is a member of the nerve growth factor family. It is induced by cortical neurons, and is necessary for survival of striatal neurons in the brain. Expression of this gene is reduced in both Alzheimer's and Huntington disease patients. This gene may play a role in the regulation of stress response and in the biology of mood disorders. Multiple transcript variants encoding distinct isoforms have been described for this gene. [provided by RefSeq, Jan 2009]
UniProt Code:	P23560
NCBI GenInfo Identifier:	114900
NCBI Gene ID:	627
NCBI Accession:	P23560.1
UniProt Secondary Accession:	P23560,Q598Q1, Q6DN19, Q6YNR2, Q6YNR3, Q9BYY7, Q9UC24 A7LA85, A7LA92, D3DQZ2,
UniProt Related Accession:	P23560
Molecular Weight:	31,116 Da
NCBI Full Name:	Brain-derived neurotrophic factor
NCBI Synonym Full Names:	brain-derived neurotrophic factor
NCBI Official Symbol:	BDNF
NCBI Official Synonym Symbols:	ANON2; BULN2
NCBI Protein Information:	brain-derived neurotrophic factor; abrineurin; neurotrophin
UniProt Protein Name:	Brain-derived neurotrophic factor
UniProt Synonym Protein Names:	Abrineurin
Protein Family:	BDNF/NT-3 growth factors receptor
UniProt Gene Name:	BDNF
UniProt Entry Name:	BDNF_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
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 clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum 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 anticoagulant. 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 & homogenize in 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.