Proteins from equal volume cell culture media samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Membranes were probed with a primary antibody against LGALS3 (sc-23983; Santa Cruz Biotech, Santa Cruz, CA) overnight (at 1:200 dilution) at 4°C with gentle shaking. Membranes were then incubated for 1 hour with secondary antibody–horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:10,000. Specific signals were developed for 1 min using the enhanced chemiluminescence (ECL) kit components 1 and 2 (GE Healthcare UK Limited, Buckinghamshire, UK). Chemiluminescence was visualized by exposure of photographic film (LAS-4000; Fujifilm, Tokyo, Japan).
Secondary antibody horseradish peroxidase conjugates
Manufactured by Santa Cruz Biotechnology
Secondary antibody–horseradish peroxidase conjugates are a type of laboratory reagent used in various immunoassay techniques. They consist of a secondary antibody that is chemically conjugated to the enzyme horseradish peroxidase. The core function of these conjugates is to provide a detectable signal in immunodetection methods, such as Western blotting and ELISA, by catalyzing a color-producing reaction when a suitable substrate is added.
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2 protocols using secondary antibody horseradish peroxidase conjugates
1
Detecting LGALS3 Protein Expression
2
Western Blot Analysis of Recombinant Proteins
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Proteins from equal volume cell culture media samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Membranes were probed with primary antibodies overnight at 4 °C with gentle shaking. Membranes were then incubated for 1 h with secondary antibody–horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:10,000. Specific signals were developed for 1 min using enhanced chemiluminescence (enhanced chemiluminescence (ECL) kit components 1 and 2, GE Healthcare UK Limited, Buckinghamshire, UK). Chemiluminescence was visualized by exposure of photographic film (LAS-4000, Fujifilm, Tokyo, Japan).
The following primary antibodies were used: a human CLU-specific antibody, which binds to the alpha-chain in the unreduced protein, (sc-6420, Santa Cruz Biotechnology, Santa Cruz, CA, USA); a strep-tag-specific antibody (IBA 2-1507-001, IBA Lifesciences, Göttingen, Germany); an his-tag-specific antibody (ab18184, Abcam, Cambridge, UK).
The following primary antibodies were used: a human CLU-specific antibody, which binds to the alpha-chain in the unreduced protein, (sc-6420, Santa Cruz Biotechnology, Santa Cruz, CA, USA); a strep-tag-specific antibody (IBA 2-1507-001, IBA Lifesciences, Göttingen, Germany); an his-tag-specific antibody (ab18184, Abcam, Cambridge, UK).
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