ADAM8 antibody (AA 145-493)

Catalog No. ABIN1866534

This anti-ADAM8 antibody is a Rabbit Polyclonal antibody detecting ADAM8 in WB, IHC, IP and ICC. Suitable for Mouse. Independently validated for use in Western Blotting.

Quick Overview for ADAM8 antibody (AA 145-493) (ABIN1866534)

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

Reactivity: Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This ADAM8 antibody is un-conjugated

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

Product Details anti-ADAM8 Antibody

Binding Specificity: AA 145-493

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

Specificity: 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.

Cross-Reactivity: Human, Rat

Purification: 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

Isotype: IgG

Application Details

Application Notes: 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.

Comment:

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: 02/11/2021

Antigen: ADAM8

Lot Number: PAA620Mu01

Method validated: Western Blotting

Positive Control:

Wildtype bone marrow derived mouse macrophages

Negative Control:

Adam8 knock-out bone marrow derived mouse macrophages

Notes:

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).

Full Methods

Primary Antibody: ABIN1866534

Secondary Antibody: 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).

Experimental 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.

Handling

Format: Liquid

Concentration: 0.5 mg/mL

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

Preservative: ProClin

Precaution of Use: 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.

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: 4 °C,-20 °C

Storage Comment: 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.

Expiry Date: 24 months

Target Details for ADAM8

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

Alternative Name: ADAM8

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

Pathways: Activation of Innate immune Response, M Phase