ADAM8 Antikörper (AA 145-493)

Produktnummer ABIN1866534

Produktdetails anti-ADAM8 Antikörper

Target: ADAM8 (ADAM Metallopeptidase Domain 8 (ADAM8))

Bindungsspezifität: AA 145-493

Reaktivität: Maus

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser ADAM8 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), Immunocytochemistry (ICC)

Verwendungszweck: Polyclonal Antibody to A Disintegrin And Metalloprotease 8 (ADAM8)

Spezifität: The antibody is a rabbit polyclonal antibody raised against ADAM8. It has been selected for its ability to recognize ADAM8 in immunohistochemical staining and western blotting.

Kreuzreaktivität: Human, Ratte

Aufreinigung: Antigen-specific affinity chromatography followed by Protein A affinity chromatography

Immunogen: Recombinant A Disintegrin And Metalloprotease 8 (ADAM8) corresdonding to Glu145~Cys493 with N-terminal His Tag

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise:

Western blotting: 0.5-5 μg/mL Immunohistochemistry: 5-20 μg/mL Immunocytochemistry: 5-20 μg/mL Optimal working dilutions must be determined by end user.

Kommentare:

The thermal stability is described by the loss rate. The loss rate was determined by accelerated thermal degradation test, that is, incubate the protein at 37°C for 48h, and no obvious degradation and precipitation were observed. The loss rate is less than 5% within the expiration date under appropriate storage condition.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #104303 (Western Blotting)

by: Department of Neurosurgery, Philipps-University Marburg

No.: #104303

Datum: 11.02.2021

Antigen: ADAM8

Chargennummer: PAA620Mu01

Validierte Anwendung: Western Blotting

Positivkontrolle:

Wildtype bone marrow derived mouse macrophages

Negativkontrolle:

Adam8 knock-out bone marrow derived mouse macrophages

Bewertung:

Passed. ABIN1866534 specifically recognizes Adam8 in lysates of bone marrow derived mouse macrophages.

Validierungsbilder

Western blot analysis of cell lysates from wildtype (WT) or Adam8 knock-out (KO) bone marrow derived mouse macrophages with ABIN1866534 (upper panels) or a beta-tubulin loading control (lower panels). Primary antibody incubation in TBST containing 5% milk (left) or 5% BSA (right).

Protokoll

Primärantikörper: ABIN1866534

Sekundärantikörper: ABIN269949

Full Protocol:

• Isolate and subsequently grow Adam8 wt or ko bone marrow derived mouse macrophages in DMEM (Gibco, 11965-092, lot 2258599) supplemented with FBS superior (Sigma Aldrich, S0615, lot 0001640840), Pen Strep (Gibco, 15140-122, lot 2240835), and Murine M-CSF (PeproTech, 315-02, lot 0419245) at 37°C and 5% CO₂ in 2 mL on a 6 well plate to 70% confluency.
• Lyse 800,000 cells in 50 µL RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 10 mM phenanthrolin, 10 mM EDTA, phosphatase and proteinase inhibitor).
• Determine total protein content of the lysates using Pierce BCA Protein Assay Kit (Thermo Scientific, 23225, lot RH237553).
• Denature 10 µg of total protein for 5 min at 95 °C in 1x Laemmli Buffer and subsequently separate them on a denaturing 10% SDS PAGE gel (polyacrylamide, Tris-HCl pH 6.8 for the stacking gel and pH 8.8 for separating gel, water, ammonium persulfate, SDS, tetramethyldiamine in a Mini-PROTEAN Tetra System (BioRad) for 1.5 h at 120 V.
• Transfer proteins onto nitrocellulose membrane (GE Healthcare Life Science, 10600001, lot A29635973) with a Western blotting system for 70 min at 150 mA.
• Block the membrane with TBST containing 5% milk or 5% BSA for 1 h at RT.
• Incubation with primary
• • rabbit anti-ADAM8 antibody (antibodies-online, ABIN1866534, PAA620Mu01) diluted 1:1,000 in TBST containing 5% milk or 5% BSA for over night at 4°C.
  • rabbit-anti TUBB antibody (Novus Biologicals, NB600-936, lot J) diluted 1:2,000 in TBST containing 5% milk over night at 4°C.
• Wash membrane 3x for 10 min with TBST.
• Incubation with secondary Donkey Anti-Rabbit IgG H&L (HRP) (abcam, ab97064, lot GR3209122-1) diluted 1:5,000 in TBST containing 5% milk for 1h at RT.
• Wash membrane 3x for 10 min with TBST.
• Reveal protein bands using Western Bright Chemilumineszenz Substrat Sirius (Biozym, 541021, no 200331-75) on a Developing machine, Royal Intas ChemoCam Imager (Inta Science Imaging) exposure time 20 sec (TBST with 5% milk) or 3 sec (TBST with 5% BSA).

Anmerkungen:

• ABIN1866534 reveals protein bands and 90 kDa and 110 kDa specifically in the Adam8 wildtype lysates. They are not present in the Adam8 ko lysates. And additional unspecific band between these two bands in visible in all lysates.

• Blocking with TBST containing 5% milk gives clearer results that blocking with TBST containing 5% BSA.

Handhabung

Format: Liquid

Konzentration: Lot specific

Buffer: 0.01M PBS, pH 7.4, containing 0.05 % Proclin-300, 50 % glycerol.

Konservierungsmittel: ProClin

Vorsichtsmaßnahmen: WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.

Handhabung: Avoid repeated freeze-thaw cycles.

Lagerung: 4 °C,-20 °C

Informationen zur Lagerung: Store at 4°C for frequent use. Stored at -20°C in a manual defrost freezer for two year without detectable loss of activity. Avoid repeated freeze-thaw cycles.

Haltbarkeit: 24 months

Details zu ADAM8

Target: ADAM8 (ADAM Metallopeptidase Domain 8 (ADAM8))

Andere Bezeichnung: A Disintegrin And Metalloprotease 8 (ADAM8 Produkte)

Synonyme: CD156 antikoerper, MS2 antikoerper, CD156a antikoerper, E430039A18Rik antikoerper, adam8 antikoerper, reprolysin antikoerper, si:zc217g15.7 antikoerper, zgc:64059 antikoerper, ADAM8 antikoerper, RGD1566394 antikoerper, ADAM metallopeptidase domain 8 antikoerper, a disintegrin and metalloproteinase domain 8 antikoerper, a disintegrin and metallopeptidase domain 8 antikoerper, ADAM metallopeptidase domain 8a antikoerper, ADAM8 antikoerper, LOC100304737 antikoerper, Adam8 antikoerper, adam8a antikoerper, adam8 antikoerper

Hintergrund: CD156-A, MS2, CD156a, Disintegrin and metalloproteinase domain-containing protein 8

Pathways: Activation of Innate immune Response, M Phase