Samples (5 μg) of the cell lysates were separated on a 15% (w/v) polyacrylamide gel 30 . Proteins were blotted onto a nitrocellulose membrane (Protran BA85, GE Healthcare UK) in a semidry blotting system (NA‐1513, Nihon Eidoh, Japan) 31 . Nitrocellulose membranes were blocked with 1% (w/v) BSA. Blocked membranes were incubated with anti‐HIF‐1α antibody for HIF‐1α (× 1000, Bethyl Laboratories, Montgomery, TX, USA), then with horseradish peroxidase (HRP)‐conjugated anti‐rabbit IgG goat antibody (Biosource, Camarillo, CA, USA). The blots were subsequently developed on a chemiluminescent detection system 32 or Imobiron (Merck Millipore, Billerica, MA, USA) using LuminoGraph (Atto, Amherst, NY, USA). The bands were densitometrically analyzed using cs analyzer software (Atto corp., Tokyo, Japan).
Imobiron
Manufactured by Merck Group
Sourced in United States, Japan
Imobiron is a laboratory equipment product developed by Merck Group. It is designed for the separation and purification of biomolecules, such as proteins, enzymes, and nucleic acids, through the process of ion exchange chromatography. The core function of Imobiron is to facilitate the efficient isolation and concentration of target analytes from complex biological samples.
Automatically generated - may contain errors
2 protocols using imobiron
1
Western Blot Analysis of HIF-1α
2
Ceramide Regulation Pathway Analysis
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The ceramide analysis performed using thin-layer membranes were incubated with anti-CerS3 rabbit antibody (1:2,000; Cusabio, Houston, TX, USA), with anti-ELOVL4 rabbit antibody (1:1,000; Cusabio, USA), or with anti-β-actin mouse monoclonal antibody (1:5,000, Wako Chemical, Tokyo, Japan). This was followed by incubation with horseradish peroxidase-conjugated antirabbit immunoglobulin G (IgG) goat antibody (1:5,000; BioSource, Camarillo, CA, USA) for CerS3 or ELOVL4, or horseradish peroxidase-conjugated anti-mouse IgG goat antibody (1:5,000; Wako Chemical) for β-actin. The blots were subsequently developed using an Immunostar LD (Wako Chemical, Japan) or Imobiron (Merck Millipore, Billerica, MA, USA) using LuminoGraph (Atto Corporation, Tokyo, Japan). The bands were densitometrically analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA).
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