MFN1 antibody (AA 1-234)

Catalog No. ABIN5013951

The Rabbit Polyclonal anti-MFN1 antibody has been validated for WB, IHC, IP and ICC and has been independently validated. It is suitable to detect MFN1 in samples from Mouse.

Quick Overview for MFN1 antibody (AA 1-234) (ABIN5013951)

Target: MFN1 (Mitofusin 1 (MFN1))

Reactivity: Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This MFN1 antibody is un-conjugated

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

Product Details anti-MFN1 Antibody

Binding Specificity: AA 1-234

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: 0.01-5 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 #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: 0.5 mg/mL

Buffer: PBS, pH 7.4, containing 0.02 % Sodium azide, 50 % glycerol.

Preservative: Sodium azide

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: MFN1

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