MFN1 antibody (AA 1-234)

Catalog No. ABIN5013951

Product Details anti-MFN1 Antibody

Target: MFN1 (Mitofusin 1 (MFN1))

Binding Specificity: AA 1-234

Reactivity: Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This MFN1 antibody is un-conjugated

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

Purpose: Polyclonal Antibody to Mitofusin 1 (MFN1)

Specificity: The antibody is a rabbit polyclonal antibody raised against MFN1. It has been selected for its ability to recognize MFN1 in immunohistochemical staining and western blotting.

Cross-Reactivity: Human, Rat

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

Immunogen: Recombinant Mitofusin 1 (MFN1) corresdonding to Met1~Ile234

Isotype: IgG

Application Details

Application Notes:

Western blotting: 0.5-2 μg/mL

1:500-2000 Immunohistochemistry: 5-20 μg/mL

1:50-200 Immunocytochemistry: 5-20 μg/mL

1:50-200 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 #103013 (Western Blotting)

by: Wang Lab, Department of Animal Science, College of Agriculture & Natural Resources

No.: #103013

Date: 05/30/2018

Antigen: MFN1

Lot Number: A20180411903

Method validated: Western Blotting

Positive Control:

293T cells transfected with MFN1-HA expression vector; the full length MFN1 CDS has been inserted upstream of an HA-tag in plasmid pcDNA3.1.

Negative Control:

293T cells transfected with an LUC-HA expression vector, and the full length LUC CDS has been inserted upstream of an HA-tag in plasmid pcDNA3.1.

293T cells transfected with MFN2-HA expression vector; the full length MFN2 CDS has been inserted upstream of an FLAG-tag in plasmid pcDNA3.1.

Notes:

Passed. ABIN5013951 specifically recognizes antigen in sample.

Validation Images

Western blot analysis of lysates from 293T cells ectopically expressing HA-tag labeled human MFN1 (MFN1-HA), luciferase (LUC-HA), and human MFN2 (MFN2-HA). Protein bands were revealed using ABIN5013951, an HA-tag antibody, an MFN2 antibody, and a beta-actin loading control.

Full Methods

Primary Antibody: ABIN5013951

Secondary Antibody: goat anti-rabbit IgG (H+L) antibody HRP conjugate (Bio-Rad, 170-6515)

Full Protocol:

• Grow 293T cells in DMEM medium (Gibco, lot 1897400) supplemented with 10% fetal bovine serum (Gibco, lot 1913756) and 1% Penicillin-Streptomycin (thermo, 10378016), at 37°C and 7% CO₂ in 6-well plates to 60-70% confluency.
• Transfect cells with plasmids expressing either MFN1-HA, MFN2-HA or LUC-HA with max 2µg of plasmid using PEI transfection reagent (Poly-Science, 24765-1) following the manufacturer´s instructions.
• Lyse cells in 100µl RIPA buffer containing protease inhibitors (Sigma-Aldrich, P8340).
• Determine total protein content of the lysates using a Bradford protein assay (Protein Assay Dye Reagent Concentrate, Bio-Rad, 5000006).
• Denature 100µg total protein for 10min at 95°C in 1x SDS loading buffer and subsequently separate them on a freshly cast 10% denaturing polyacrylamide gel.
• Transfer proteins onto Immun-Blot Low Fluorescence PVDF Membrane (0.45μm) at 1.0A and up to 25V for 30min via Trans-Blot Turbo Transfer System.
• Block the membrane with Bio-rad Blotting-Grade Blocker (5% non-fat milk in PBS).
• Incubation with primary
• • rabbit anti-MFN1 antibody (antibodies-online, ABIN5013951, lot A20180411903) diluted 1:200 in Blocking Buffer (5% non-fat milk) ON at 4°C.
  • mouse anti-HA-tag antibody (Santa Cruz, sc-7392) diluted 1:2000 in Blocking Buffer (5% non-fat milk) ON at 4°C (only LUC-HA expressing cells).
  • mouse anti-MFN2-tag antibody (Santa Cruz, sc-100560) diluted 1:2000 in Blocking Buffer (5% non-fat milk) ON at 4°C.
  • mouse anti-beta-actin antibody (Sigma-Aldrich, A1978) diluted 1:10000 in Blocking Buffer(5% non-fat milk) ON at 4°C.
• Wash membrane 3x for 10min with PBS supplemented with 0.1% Tween 20.
• Incubation with secondary goat anti-rabbit IgG (H+L) antibody HRP conjugate (Bio-Rad, 170-6515) or goat anti-mouse IgG (H+L) antibody HRP conjugate (Bio-Rad, 170-6516) diluted 1:10000 in Odyssey Blocking Buffer for 1h at RT.
• Wash membrane 3x for 10min with PBS supplemented with 0.1% Tween 20.
• Add Clarity and Clarity Max Western ECL Blotting Substrates and scan the membrane on a Bio-Rad ChemiDoc XRS+System.

Experimental Notes:

The antigen antibody ABIN5013951 reveals a protein of the expected molecular weight of antigen in lysates of cells. The protein bands is only visible in the positive but not the negative controls.

Handling

Format: Liquid

Concentration: Lot specific

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

Preservative: ProClin

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 MFN1

Target: MFN1 (Mitofusin 1 (MFN1))

Alternative Name: Mitofusin 1 (MFN1 Products)

Synonyms: mfn1 antibody, zgc:66150 antibody, mitofusin-1 antibody, MFN1 antibody, MFN2 antibody, hfzo1 antibody, hfzo2 antibody, 2310002F04Rik antibody, 6330416C07Rik antibody, D3Ertd265e antibody, HR2 antibody, mKIAA4032 antibody, Fzo1b antibody, mitofusin 1b antibody, mitofusin 1 antibody, mitofusin 1 S homeolog antibody, mfn1b antibody, MFN1 antibody, mfn1.S antibody, mfn1 antibody, Mfn1 antibody

Background: Hfzo1, Hfzo2, Transmembrane GTPase MFN1, Fzo homolog