Human Proprotein Convertase 9/PCSK9 DuoSet ELISA

R&D Systems | Catalog # DY3888

R&D Systems
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Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

125-8000 pg/mL

Sample Type

Cell culture supernates, serum, and plasma
Note: Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet

Reactivity

Human

Human Proprotein Convertase 9/PCSK9 DuoSet ELISA Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits
View all Proprotein Convertase 9/PCSK9 ELISA Kits »

Product Summary for Human Proprotein Convertase 9/PCSK9 DuoSet ELISA

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Proprotein Convertase 9/PCSK9. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Product Specifications

Assay Format

96-well strip plate (sold separately)

Sample Volume Required

100 µL

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Biotin

Specificity

Please see the product datasheet

Label

HRP

Scientific Data Images for Human Proprotein Convertase 9/PCSK9 DuoSet ELISA

Human Proprotein Convertase 9 / PCSK9 ELISA Standard Curve

Human Proprotein Convertase 9 / PCSK9 ELISA Standard Curve

Kit Contents for Human Proprotein Convertase 9/PCSK9 DuoSet ELISA

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

The components listed above may be purchased separately:

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4

Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: R&D Systems (Catalog # DY990)

Plate Sealers: ELISA Plate Sealers (Catalog # DY992)

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: Proprotein Convertase 9/PCSK9

Proprotein convertase subtilisin kexin 9 (PCSK9), also named neural apoptosis-regulated convertase 1 (NARC-1), is a member of the proteinase K subfamily of subtilisin-related serine endoproteases. The full-length protein has 692 amino acids, including a signal peptide, a pro- domain, and a catalytic domain. PCSK9 is highly expressed in the liver, intestine, and kidney. It is initially synthesized as a soluble 74 kDa precursor protein. In the endoplasmic reticulum, it undergoes autocatalytic intramolecular cleavage to generate a 14 kDa pro- domain and a 60 kDa catalytic domain. These two domains remain associated when PCSK9 is secreted outside the cells (1-3). The primary physiologic function of PCSK9 is to mediate the degradation of low density lipoprotein receptor (LDL R). Early observations indicated that gain-of-function missense mutations in the PCSK9 gene can cause an autosomal dominant form of hypercholesterolemia (4, 5). The expression of PCSK9 was observed to be up-regulated by the sterol regulatory element binding proteins (SREBPs), a family of transcription factors that are responsible for the upregulation of genes involved in cholesterol and fatty acid metabolism, such as the LDL R gene (6, 7). Further experimental evidence revealed that in mice, when the PCSK9 gene was knocked out, the number of LDL R in hepatocytes increased, whereas when PCSK9 was over-expressed, the amount of LDL R protein was reduced in the liver (8, 9). In humans, genetic analyses have shown that individuals who have nonsense or loss-of-function mutations in the PCSK9 gene have significantly lower plasma LDL cholesterol levels (10, 11). These investigations clearly indicated that PCSK9 plays a key role in reducing the hepatic LDL R levels. Recently, the underlying mechanism has been uncovered: under normal physiologic conditions, the LDL R is internalized on the cell surface and directed to the endosomes in order to be recycled back to the cell surface. PCSK9 binds to the EGF domain of the LDL R and prevents LDL R from being sorted to the endosomes. Instead, the PCSK9/LDL R complex is redistributed to the lysosomes for degradation (12-14). As such, PCSK9 regulates the amount of LDL R in the circulation and modulates cholesterol levels. Serum PCSK9 concentrations have been found to be directly associated with cholesterol levels (15, 16). Since individuals with loss-of-function PCSK9 mutations have strikingly reduced risk of coronary heart diseases, PCSK9 has become an attractive drug target in recent years (17, 18). One approach is to generate small molecules that are able to interfere with PCSK9 autoactivation and its interaction with LDL R. Other approaches aiming to reduce the amounts of PCSK9 in the circulation, such as small interfering RNAs (siRNAs), have also shown promise (19, 20).

Alternate Names

FH3, FHCL3, HCHOLA3, LDLCQ1, NARC-1, NARC1, PC9, PCSK9

Entrez Gene IDs

255738 (Human); 100102 (Mouse); 298296 (Rat); 102142788 (Cynomolgus Monkey)

Gene Symbol

PCSK9

Additional Proprotein Convertase 9/PCSK9 Products

Product Documents for Human Proprotein Convertase 9/PCSK9 DuoSet ELISA

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Human Proprotein Convertase 9/PCSK9 DuoSet ELISA

For research use only

Related Research Areas

Obesity
Serine Proteases and Regulators

Citations for Human Proprotein Convertase 9/PCSK9 DuoSet ELISA

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  • Human Proprotein Convertase 9/PCSK9 DuoSet ELISA
    Name: Anonymous
    Sample Tested: EDTA Plasma
    Verified Customer | Posted 09/25/2024
    EDTA plasma, human, diluted 1:100, beautiful!
    Human Proprotein Convertase 9/PCSK9 DuoSet ELISA DY3888
  • Human Proprotein Convertase 9/PCSK9 DuoSet ELISA
    Name: Anonymous
    Sample Tested: Serum and Serum
    Verified Customer | Posted 07/05/2023
    Human Proprotein Convertase 9/PCSK9 DuoSet ELISA DY3888
  • Human Proprotein Convertase 9/PCSK9 DuoSet ELISA
    Name: Anonymous
    Sample Tested: Plasma
    Verified Customer | Posted 05/22/2023
    Human Proprotein Convertase 9/PCSK9 DuoSet ELISA DY3888
  • Human Proprotein Convertase 9/PCSK9 DuoSet ELISA
    Name: Christian Cassel
    Sample Tested: Plasma
    Verified Customer | Posted 08/21/2019

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Protocols

View specific protocols for Human Proprotein Convertase 9/PCSK9 DuoSet ELISA (DY3888):

GENERAL ELISA PROTOCOL

Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

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