Human PCSK9/Proprotein Convertase 9 ELISA Kit

Product Name:	Human PCSK9/Proprotein Convertase 9 ELISA Kit
Product Code:	HUFI00222
Size:	96 Assays
Alias:	PCSK9, Proprotein Convertase Subtilisin, Kexin Type 9, Proprotein Convertase 9, FH3, HCHOLA3, NARC-1, PC9, HCHOLA3, LDLCQ1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PCSK9 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.375ng/ml
Range:	0.625-40ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PCSK9 and the recovery rates were calculated by comparing the measured value to the expected amount of Human PCSK9 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-97	92
EDTA plasma(n=5)	87-105	95
UFH plasma(n=5)	91-102	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PCSK9 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%	88-105%	86-105%
EDTA plasma(n=5)	84-100%	85-98%	84-101%
UFH plasma(n=5)	86-97%	81-95%	80-98%

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

Q8NBP7

UniProt Protein Function:	PCSK9: Crucial player in the regulation of plasma cholesterol homeostasis. Binds to low-density lipid receptor family members: low density lipoprotein receptor (LDLR), very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1/APOER) and apolipoprotein receptor 2 (LRP8/APOER2), and promotes their degradation in intracellular acidic compartments. Acts via a non-proteolytic mechanism to enhance the degradation of the hepatic LDLR through a clathrin LDLRAP1/ARH-mediated pathway. May prevent the recycling of LDLR from endosomes to the cell surface or direct it to lysosomes for degradation. Can induce ubiquitination of LDLR leading to its subsequent degradation. Inhibits intracellular degradation of APOB via the autophagosome/lysosome pathway in a LDLR-independent manner. Involved in the disposal of non-acetylated intermediates of BACE1 in the early secretory pathway. Inhibits epithelial Na(+) channel (ENaC)-mediated Na(+) absorption by reducing ENaC surface expression primarily by increasing its proteasomal degradation. Regulates neuronal apoptosis via modulation of LRP8/APOER2 levels and related anti-apoptotic signaling pathways. Defects in PCSK9 are the cause of hypercholesterolemia autosomal dominant type 3 (HCHOLA3). A familial condition characterized by elevated circulating cholesterol contained in either low-density lipoproteins alone or also in very-low-density lipoproteins. Belongs to the peptidase S8 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted, signal peptide; EC 3.4.21.-; Protease; Secreted; Cell development/differentiation Chromosomal Location of Human Ortholog: 1p32.3 Cellular Component: Golgi apparatus; extracellular space; cell surface; rough endoplasmic reticulum; lysosome; endoplasmic reticulum; early endosome; ER to Golgi transport vesicle; extrinsic to external side of plasma membrane; perinuclear region of cytoplasm; cytoplasm; late endosome; plasma membrane Molecular Function:sodium channel inhibitor activity; very-low-density lipoprotein binding; protein binding; protein self-association; low-density lipoprotein receptor binding; serine-type endopeptidase activity; low-density lipoprotein binding; apolipoprotein binding; apolipoprotein receptor binding Biological Process: cholesterol metabolic process; lysosomal transport; apoptosis; positive regulation of receptor internalization; lipoprotein metabolic process; regulation of low-density lipoprotein receptor catabolic process; liver development; cellular response to starvation; proteolysis; neuron differentiation; protein autoprocessing; cholesterol homeostasis; triacylglycerol metabolic process; cellular response to insulin stimulus; neurogenesis; phospholipid metabolic process; positive regulation of neuron apoptosis; regulation of neuron apoptosis; negative regulation of receptor recycling; low-density lipoprotein receptor catabolic process; regulation of receptor activity; kidney development Disease: Hypercholesterolemia, Autosomal Dominant, 3
NCBI Summary:	This gene encodes a member of the subtilisin-like proprotein convertase family, which includes proteases that process protein and peptide precursors trafficking through regulated or constitutive branches of the secretory pathway. The encoded protein undergoes an autocatalytic processing event with its prosegment in the ER and is constitutively secreted as an inactive protease into the extracellular matrix and trans-Golgi network. It is expressed in liver, intestine and kidney tissues and escorts specific receptors for lysosomal degradation. It plays a role in cholesterol and fatty acid metabolism. Mutations in this gene have been associated with autosomal dominant familial hypercholesterolemia. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Feb 2014]
UniProt Code:	Q8NBP7
NCBI GenInfo Identifier:	317373487
NCBI Gene ID:	255738
NCBI Accession:	Q8NBP7.3
UniProt Secondary Accession:	Q8NBP7,Q5PSM5, Q5SZQ2, A8T640, C0JYY9,
UniProt Related Accession:	Q8NBP7
Molecular Weight:	20,827 Da
NCBI Full Name:	Proprotein convertase subtilisin/kexin type 9
NCBI Synonym Full Names:	proprotein convertase subtilisin/kexin type 9
NCBI Official Symbol:	PCSK9
NCBI Official Synonym Symbols:	FH3; PC9; NARC1; LDLCQ1; NARC-1; HCHOLA3
NCBI Protein Information:	proprotein convertase subtilisin/kexin type 9; subtilisin/kexin-like protease PC9; neural apoptosis regulated convertase 1; convertase subtilisin/kexin type 9 preproprotein
UniProt Protein Name:	Proprotein convertase subtilisin/kexin type 9
UniProt Synonym Protein Names:	Neural apoptosis-regulated convertase 1; NARC-1; Proprotein convertase 9; PC9; Subtilisin/kexin-like protease PC9
Protein Family:	Proprotein convertase subtilisin/kexin
UniProt Gene Name:	PCSK9
UniProt Entry Name:	PCSK9_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.

Human PCSK9/Proprotein Convertase 9 ELISA Kit (HUFI00222)

Description