ADAM8 anticorps (AA 145-493)

N° du produit ABIN1866534

Cet anticorps anti-ADAM8 est un anticorps Lapin Polyclonal détectant ADAM8 dans WB, IHC, IP et ICC. Adapté pour Souris. Validé indépendamment pour une utilisation dans Western Blotting.

Aperçu rapide pour ADAM8 anticorps (AA 145-493) (ABIN1866534)

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

Reactivité: Souris

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Cet anticorp ADAM8 est non-conjugé

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

Détail du produit anti-ADAM8 anticorps

Épitope: AA 145-493

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

Specificité: 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.

Réactivité croisée: Humain, Rat

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

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

Isotype: IgG

Information d'application

Indications d'application: Western blotting: 0.01-5 μg/mL,Immunohistochemistry: 5-20 μg/mL,Immunocytochemistry: 5-20 μg/mL,Optimal working dilutions must be determined by end user.

Commentaires:

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.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #104303 (Western Blotting)

by: Department of Neurosurgery, Philipps-University Marburg

No.: #104303

Date: 11.02.2021

Antigène: ADAM8

Numéro du lot: PAA620Mu01

Application validée: Western Blotting

Contrôle positif:

Wildtype bone marrow derived mouse macrophages

Contrôle négative:

Adam8 knock-out bone marrow derived mouse macrophages

Conclusion:

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

Validation Images

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).

Protocole

Anticorps primaire: ABIN1866534

Anticorps secondaire: 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).

Notes:

• 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.

Stockage

Format: Liquid

Concentration: 0.5 mg/mL

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

Agent conservateur: ProClin

Précaution d'utilisation: 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.

Conseil sur la manipulation: Avoid repeated freeze-thaw cycles.

Stock: 4 °C,-20 °C

Stockage commentaire: 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.

Date de péremption: 24 months

Détails sur ADAM8

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

Autre désignation: ADAM8

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

Pathways: Activation of Innate immune Response, M Phase