Human P4HB ELISA Pair Set

Details zu Produkt Nr. ABIN2010471, Anbieter: Anmelden zum Anzeigen
  • DSI
  • ERBA2L
  • GIT
  • P4Hbeta
  • PDI
  • PDIA1
  • PHDB
  • PO4DB
  • PO4HB
  • ERp59
  • Pdia1
  • Thbp
  • PDIR
  • p55
  • p58
  • prolyl 4-hydroxylase subunit beta
  • prolyl 4-hydroxylase, beta polypeptide
  • prolyl 4-hydroxylase subunit beta L homeolog
  • protein disulfide-isomerase
  • P4HB
  • P4hb
  • p4hb.L
  • LOC8281065
Sandwich ELISA
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Verwendungszweck The Human P4HB ELISA Pair Set is for the quantitative determination of Human P4HB.
Nachweismethode Colorimetric
Sensitivität 47 pg/mL
Produktmerkmale Capture Antibody: 1 mg/mL of mouse anti-Human P4HB monoclonal antibody (in PBS, pH 7.4). Dilute to a working concentration of 2 μg/mL in PBS before coating.

Detection Antibody: 0.2 mg/mL of mouse anti-Human P4HB monoclonal antibody conjugated to horseradish-peroxidase (HRP) (in PBS, 50 % HRP-Protector, pH 7.4, store at 4 °C). Dilute to working concentration of 1 μg/mL in detection antibody dilution buffer before use.

Standard: Each vial contains 85 ng of recombinant Human P4HB. Reconstitute with 1 mL detection antibody dilution buffer. After reconstitution, store at -20 °C to -80 °C in a manual defrost freezer. A seven-point standard curve using 2-fold serial dilutions in sample dilution buffer, and a high standard of 3000 pg/mL is recommended.

Sensitivity: The minimum detectable dose of Human P4HB was determined to be approximately 47 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
  • Capture Ab
  • Detection Ab
  • Standard
Benötigtes Material CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Andere Bezeichnung P4HB
Hintergrund Prolyl 4-hydroxylase belongs to the group of 2-oxoglutarate coupled dioxygenases, and catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. The reaction product, thegenetically uncoded amino acid 4-hydroxyproline, plays a crucial role in collagens, as the hydroxyl groups of theseamino acid residues are essential for the folding of the newly synthesized procollagen polypeptide chains into triple-helical molecules. The active enzyme is a tetramer (α2β2) , and the β subunit (P4HB) alnoe has been found to beidentical to the enzyme protein disulfide isomerase (PDI) . P4HB is multifunctional protein catalyzes the formation, breakage and rearrangement of disulfide bonds. Inside the cell, P4HB functions as a chaperone that inhibitsaggregation of misfolded proteins at high concentrations, while facilitates aggregation (anti-chaperone activity) at lowconcentrations. In addition to a subunit of polyl 4-hydroxylase, P4HB also acts as a structure subunit of microsomaltriacylglycerol transfer protein MTTP.
Applikationshinweise Optimal working dilution should be determined by the investigator.
Protokoll This ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes a monoclonal antibody specific for Human P4HB coated on a 96-well plate. Standards and samples are added to the wells, and any Human P4HB present binds to the immobilized antibody. The wells are washed and a horseradish peroxidase conjugated mouse anti-Human P4HB monoclonal antibody is then added, producing an antibody-antigen-antibody "sandwich". The wells are again washed and TMB substrate solution is loaded, which produces color in proportion to the amount of Human P4HB present in the sample. To end the enzyme reaction, the stop solution is added and absorbances of the microwell are read at 450 nm.
Aufbereitung der Reagenzien
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer 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.
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    6. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
    7. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.

Beschränkungen Nur für Forschungszwecke einsetzbar
Format Liquid
Handhabung Avoid repeated freeze-thaw cycles.
Lagerung 4 °C,-20 °C,-80 °C
Informationen zur Lagerung Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Avoid repeated freeze-thaw cycles.

Detection Antibody: Store at 4°C and protect it from prolonged exposure to light for up to 6 months from date of receipt. DO NOT FREEZE!

Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month. Avoid repeated freeze-thaw cycles.
Haltbarkeit 6 months
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