UCP1 antibody (AA 179-296)

Catalog No. ABIN2905048

This anti-UCP1 antibody is a Rabbit Polyclonal antibody detecting UCP1 in WB, IHC, IP and IF/ICC. Suitable for Mouse. Independently validated for use in Western Blotting.

Quick Overview for UCP1 antibody (AA 179-296) (ABIN2905048)

Target: UCP1 (Uncoupling Protein 1 (Mitochondrial, Proton Carrier) (UCP1))

Reactivity: Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This UCP1 antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), Immunofluorescence (fixed cells) (IF/ICC)

Product Details anti-UCP1 Antibody

Binding Specificity: AA 179-296

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

Immunogen: UCP1 (Pro179-Leu296)

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #103357 (Western Blotting)

by: Mivechi lab, Georgia Cancer Center, Augusta University

No.: #103357

Date: 10/03/2018

Antigen: UCP1

Lot Number: AB1808U769

Method validated: Western Blotting

Positive Control:

Cold exposed mouse brown adipose tissue (BAT)

Negative Control:

Mouse liver tissue

Notes:

Passed. ABIN3056733 can detect mouse UCP1 protein in immunoblotting analysis.

Validation Images

Lysates from mitochondrial fraction isolated from mouse liver tissue (negative control) or brown adipose tissue (BAT) without (22°C) or with (4°C) cold exposure for 6h were resolved by SDS-PAGE gel electrophoresis followed by Western blotting analysis using UCP1 primary antibody ABIN3056733.

Full Methods

Primary Antibody: ABIN3056733

Secondary Antibody: donkey anti-rabbit HRP-conjugated antibody (Jackson Immuno Research, 711-035-152)

Full Protocol:

• Homogenize mouse brown fat tissue in 2ml mitochondria isolation buffer (70mM sucrose, 210mM mannitol, 5mM HEPES, 1mM EGTA, pH7.2, and 0.5% BSA).
• Centrifuge the homogenates for 10min at 800g at 4°C.
• Centrifuge the supernatant again at 8000g for 10min at 4°C.
• Lyse the resulting mitochondria fraction in RIPA buffer containing protease inhibitor cocktail (Pierce Protease Inhibitor, A32965, lot TA2492391).
• Determined protein concentration with Pierce BCA Protein Assay Kit (Pierce, 23225, lot OA180049).
• Separate 50µg of total protein per lane on a denaturing 12% SDS-PAGE gel (Bio-Rad, 1610156, lot 64144809) in a Mini Protean Tetra Cell for 2h at 80V.
• Transfer proteins onto Immobilon-P PVDF Membrane (Millipore, IPVH00010) using Mini Trans Blot Cell system (Bio-Rad, 1703930).
• Block membrane with 5% milk in PBS-T for 1h at RT.
• Incubate membrane with primary rabbit anti-UCP1 antibody (antibodies-online, ABIN3056733, lot AB1808U769) diluted to 1µg/ml ON at 4°C.
• Wash membrane 3x for 10min with PBS-T.
• Incubate membrane with secondary donkey anti-rabbit HRP-conjugated antibody (Jackson Immuno Research, 711-035-152) for 1h at RT.
• Wash membrane 3x for 10min with PBS-T.
• Incubate membrane Pierce ECL Western Blotting Substrate working solution (Thermo Scientific, 1859701, lot TB263310) for 1min at RT. Expose membrane to x-ray film for different amount of time.

Experimental Notes:

The UCP1 antibody ABIN3056733 reveals a specific protein band in positive control samples (cold exposed mouse brown fat tissue), but not in negative control samples (liver tissue).

Handling

Format: Liquid

Concentration: 500 μg/mL

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

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Store at 4 °C for frequent use. Aliquot and store at -20 °C for one year. Avoid repeated freeze/thaw cycles.

Target Details for UCP1

Target: UCP1 (Uncoupling Protein 1 (Mitochondrial, Proton Carrier) (UCP1))

Alternative Name: Uncoupling Protein 1, Mitochondrial

UniProt: P12242

Pathways: Proton Transport, Brown Fat Cell Differentiation