Human FBN/Fibrillin-1 ELISA Kit- High Sensitivity

Product Name:	Human FBN/Fibrillin-1 ELISA Kit
Product Code:	HUFI00739
Size:	96 Assays
Alias:	FBN1, Fibrillin-1, FBN
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human FBN1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human FBN1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human FBN1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	94-105	97
EDTA plasma(n=5)	87-103	99
UFH plasma(n=5)	86-104	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human FBN1 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)	88-105%	86-104%	95-105%
EDTA plasma(n=5)	83-99%	85-100%	82-101%
UFH plasma(n=5)	82-96%	83-95%	83-97%

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

P35555

UniProt Protein Function:	FBN1: a structural component of the 10-12 nm diameter microfibrils of the extracellular matrix, which conveys both structural and regulatory properties to load-bearing connective tissues. FBN1-containing microfibrils provide long-term force bearing structural support. In tissues such as the lung, blood vessels and skin, microfibrils form the periphery of the elastic fiber, acting as a scaffold for the deposition of elastin. In addition, microfibrils can occur as elastin-independent networks in tissues such as the ciliary zonule, tendon, cornea and glomerulus where they provide tensile strength and have anchoring roles. Plays a key role in tissue homeostasis through specific interactions with growth factors, such as the bone morphogenetic proteins (BMPs), growth and differentiation factors and latent transforming growth factor-beta-binding proteins, cell-surface integrins and other extracellular matrix protein and proteoglycan components. Regulates osteoblast maturation by controlling TGF-beta bioavailability and calibrating TGF-beta and BMP levels, respectively. Negatively regulates osteoclastogenesis by binding and sequestering an osteoclast differentiation and activation factor TNFSF11. This leads to disruption of TNFSF11-induced Ca2+ signaling and impairment of TNFSF11-mediated nuclear translocation and activation of transcription factor NFATC1 which regulates genes important for osteoclast differentiation and function. Mediates cell adhesion via its binding to cell surface receptors integrins ITGB3 and ITGB1. Binds heparin and this interaction has an important role in the assembly of microfibrils. Cleavage of N- and C-termini is required for incorporation into the extracellular matrix and assembly into microfibrils. Its cleaved C-terminal peptide (2732 ¿ 2871) is the hormone asprosin, secreted by white adipose tissue that targets the liver, stimulating the release of glucose and increasing plasma glucose levels. Acts in response to fasting, and promotes hepathocyte glucose release via a PKA-dependent pathway.
UniProt Protein Details:	Protein type:Extracellular matrix; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 15q21.1 Cellular Component: basement membrane; extracellular matrix; extracellular region; extracellular space; microfibril; proteinaceous extracellular matrix Molecular Function:calcium ion binding; extracellular matrix constituent conferring elasticity; extracellular matrix structural constituent; heparin binding; integrin binding; protein binding; protein complex binding Biological Process: camera-type eye development; cell adhesion mediated by integrin; embryonic eye morphogenesis; extracellular matrix disassembly; extracellular matrix organization and biogenesis; heart development; negative regulation of osteoclast differentiation; post-embryonic eye morphogenesis; skeletal development Disease: Acromicric Dysplasia; Ectopia Lentis 1, Isolated, Autosomal Dominant; Geleophysic Dysplasia 2; Marfan Lipodystrophy Syndrome; Marfan Syndrome; Mass Syndrome; Stiff Skin Syndrome; Weill-marchesani Syndrome 2
NCBI Summary:	This gene encodes a member of the fibrillin family of proteins. The encoded preproprotein is proteolytically processed to generate two proteins including the extracellular matrix component fibrillin-1 and the protein hormone asprosin. Fibrillin-1 is an extracellular matrix glycoprotein that serves as a structural component of calcium-binding microfibrils. These microfibrils provide force-bearing structural support in elastic and nonelastic connective tissue throughout the body. Asprosin, secreted by white adipose tissue, has been shown to regulate glucose homeostasis. Mutations in this gene are associated with Marfan syndrome and the related MASS phenotype, as well as ectopia lentis syndrome, Weill-Marchesani syndrome, Shprintzen-Goldberg syndrome and neonatal progeroid syndrome. [provided by RefSeq, Apr 2016]
UniProt Code:	P35555
NCBI GenInfo Identifier:	311033452
NCBI Gene ID:	2200
NCBI Accession:	P35555.3
UniProt Secondary Accession:	P35555,Q15972, Q75N87, B2RUU0, D2JYH6,
UniProt Related Accession:	P35555
Molecular Weight:	312,237 Da
NCBI Full Name:	Fibrillin-1
NCBI Synonym Full Names:	fibrillin 1
NCBI Official Symbol:	FBN1
NCBI Official Synonym Symbols:	FBN; SGS; WMS; MASS; MFLS; MFS1; OCTD; SSKS; WMS2; ACMICD; ECTOL1; GPHYSD2
NCBI Protein Information:	fibrillin-1
UniProt Protein Name:	Fibrillin-1
Protein Family:	Fibrillin
UniProt Gene Name:	FBN1

*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.