Thyroperoxidase Protein

Produktnummer ABIN934717

Produktdetails

Target: Thyroperoxidase (TPO) (Thyroid Peroxidase (TPO))

Protein-Typ: Native

Spezies: Human

Quelle: Human

Produktmerkmale: Purified native Human Thyroid Peroxidase protein
Protein Source: Human thyroid glands

Reinheit: > 95 % pure

Anwendungsinformationen

Applikationshinweise: Each Investigator should determine their own optimal working dilution for specific applications.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (SDS)

Validierung #102793 (SDS-PAGE)

by: in.vent Diagnostica GmbH, Hennigsdorf, Germany

No.: #102793

Datum: 17.04.2018

Antigen: TPO

Chargennummer: A17091521

Validierte Anwendung: SDS-PAGE

Positivkontrolle:

Molecular weight (Uniprot P07202: 8 possible isoforms of TPO with a molecular weight between 103 and 67 kDa)

Negativkontrolle:

none

Bewertung:

Passed. Native human TPO protein ABIN934717 appears as a band with a molecular weight of approximately 100kDa after separation on an SDS-PAGE gel.

Validierungsbilder

Coomassie staining subsequently to SDS-PAGE separation of human TPO1 ABIN934717.

Standard curve and ABIN934717 measurements using LIA.

Protokoll

Primärantikörper: ABIN934717

Full Protocol:

• Dilute 2µg of native human TPO (antibodies-online, ABIN934717, lot A17091521) in 2x sample buffer (126mM Tris-HCl pH6.8, 20% glycerol, 4% SDS, 0.02% bromophenol blue, 0.1M DTT).
• Boil samples for 5min at 95°C.
• Separate the sample on a denaturing SERVAGel TG PRiME 12% precast gel (Serva, 43266, V170144) in a Electrophoresis chamber (Hoefer Inc., SE250) with Laemmli buffer for 1h at 250V, 50mA/gel along side 4µl Roti-Mark Tricolor (Carl Roth, 8271, lot 326248051) molecular weight marker.
• Staining of the separated proteins with InstantBlue Protein Stain (Expedeon, ISB1L, lot 170616330) according to the manufacturer’s recommendations.
• Image acquisition: 600dpi scan and increasing of color saturation.

Anmerkungen:

The manufacturer’s information concerning the protein’s purity and molecular weight can be confirmed.

Independent Validation (LIA)

Validierung #103248 (Lipid Interaction Assay)

by: in.vent Diagnostica GmbH, Hennigsdorf, Germany

No.: #103248

Datum: 17.04.2018

Antigen: TPO

Chargennummer: A17091521

Validierte Anwendung: Lipid Interaction Assay

Positivkontrolle:

Protein concentration (manufacturer's specification)

Standards and controls for LIA based of purified TPO out of human thyroid

Negativkontrolle:

Dilution Buffer for LIA

Deionized water for absorbance at 280nm

Bewertung:

Passed. Protein concentration of native human TPO protein ABIN934717 can be confirmed by absorbance at 280nm and by LIA.

Validierungsbilder

Coomassie staining subsequently to SDS-PAGE separation of human TPO1 ABIN934717.

Standard curve and ABIN934717 measurements using LIA.

Protokoll

Primärantikörper: ABIN934717

Full Protocol:

• Measuring protein concentration using absorbance at 280nm:
• • 100µl sample in a 96-Well UV Microplate (Thermo Fisher Scientific, 8404)
  • Measuring at 280nm
  • Calculation with d of 0.28cm
  • Extinction coefficient assuming all pairs of Cys residues form cysteines: Abs 0.1% (=1g/l)= 1.304
• Measuring TPO concentration using an in-house coated tube LIA:
• • Sample dilution with assay dilution buffer in 6 steps (10000ng/ml and 100ng/ml as pre-dilution; 50, 25, 12.5, 6.25ng/ml as samples)
  • Pipette 300µl tracer and 200µl standards, controls, or samples per tube in duplicates.
  • Incubation ON at RT.
  • Add 1ml washing solution to each coated tube prior to decanting off the liquid.
  • Add 1 ml washing solution to each coated tube three times and decant off the liquid. Turn the tubes upside down for 5–10min and adsorb remaining liquid with blotting paper.
  • Measurement in a luminometer by automatic injection of LIA reagents (sodium hydrate, azotic acid). Measurement time: 1sec.
• Calculate the concentration based on the standard curve.

Anmerkungen:

• The LIA measures human TPO in serum, plasma and other fluids, using one anti-TPO antibody at solid phase and another anti-TPO antibody as tracer. The measurement range is from 10 to 50ng/ml.

• Absorbance at 280nm was 0.22. The calculated concentration considering the extinction coefficient is 0.6mg/ml (expected was 0.5mg/ml).

• Regression of TPO LIA standard curve was R²=0.997. Calculated concentration of LIA controls was 28ng/ml (expected 20-30ng/ml) and 14ng/ml (expected 10-15ng/ml).

• The calculated concentration of the diluted samples was 24ng/ml (expected 25ng/ml) and 13ng/ml (expected 12.5ng/ml). The other dilutions were out of measurement range.

Handhabung

Format: Lyophilized

Rekonstitution: Reconstitute in distilled water.

Buffer: Lyophilized in 20 mM TRIS, with 50 mM KCl, no preservatives.

Konservierungsmittel: Without preservative

Handhabung: Avoid repeated freeze/thaw cycles.

Lagerung: -20 °C

Informationen zur Lagerung: Aliquot and store at -20 °C.

Referenzen

Ali, Wolfe, Mukherjee, Radford, Patel, White, Milojevic, Capretta, Nair, Brennan: "A sputum bioassay for airway eosinophilia using an eosinophil peroxidase aptamer." in: Scientific reports, Vol. 12, Issue 1, pp. 22476, (2022) (PubMed).

Rahnama, Mahmoudi, Kazemnejad, Salehi, Ghahiri, Soltanghoraee, Vafaei, Rezaei, Zarnani: "Thyroid peroxidase in human endometrium and placenta: a potential target for anti-TPO antibodies." in: Clinical and experimental medicine, (2020) (PubMed).

Antigendetails

Target: Thyroperoxidase (TPO) (Thyroid Peroxidase (TPO))

Andere Bezeichnung: Thyroid Peroxidase (TPO Produkte)

Synonyme: MSA Protein, TDH2A Protein, TPX Protein, TPO Protein, msa Protein, tpx Protein, xtpo Protein, LOC100218312 Protein, thyroid peroxidase Protein, TPO Protein, Tpo Protein, tpo Protein, LOC100547103 Protein

Hintergrund: Thyroid peroxidase or thyroperoxidase (TPO) is an enzyme expressed mainly in the thyroid that liberates iodine for addition onto tyrosine residues on thyroglobulin for the production of thyroxine (T4) or triiodothyronine (T3), thyroid hormones. In humans, thyroperoxidase is encoded by the TPO gene.
Description: Human thyroid glands.
Alternative Names: TPO protein, MSA protein, TPX protein

Pathways: Thyroid Hormone Synthesis