Anti mouse igg horseradish peroxidase conjugate

Immunoblot analysis was performed as previously described [20 (link)], with a few modifications. GMC101 worms were weighed and homogenized in 500 µL of phosphate-buffered saline. The homogenates were centrifuged at 800× g for 10 min, and the supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting. SDS–PAGE was performed using p-PAGEL slab gels (P–T16.5S; ATTO Corporation, Tokyo, Japan) according to the manufacturer’s instructions.
The gels were then stained with Coomassie Brilliant Blue R-250, and proteins were transferred to polyvinylidene difluoride membranes (Immuno-Blot PVDF; Bio-Rad Laboratories, Hercules, CA, USA) using an electroblotting apparatus (model 200/2.0, Bio-Rad Laboratories) set at 13 V for 30 min. Aβ peptide was detected using a monoclonal anti-Aβ_1–16 primary antibody (BioLegend, San Diego, CA, USA) and an anti-mouse IgG–horseradish peroxidase conjugate (Promega Corp. Madison, WI, USA) secondary antibody. Tubulin (the loading control) was detected using a monoclonal anti-tubulin primary antibody (ab6160, Abcam, Cambridge, MA, USA) and an anti-mouse IgG–horseradish peroxidase conjugate (SA00001-15, Proteintech Japan, Tokyo, Japan) secondary antibody. Signals were detected using EzWestBlue (ATTO Corporation) according to the manufacturer’s instructions.

Reagents were purchased from the following sources: middle range molecular weight protein markers, anti-mouse IgG horseradish peroxidase conjugate, 5-bromo-4-chloro-3 indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), Promega; anti-rabbit IgG alkaline phosphatase conjugate, anti-bovine IgG horseradish peroxidase conjugate, anti-bovine IgG alkaline phosphatase conjugate, anti-equine IgG alkaline phosphatase conjugate, anti-mouse IgG alkaline phosphatase conjugate, diaminobenzidine (DAB), horseradish peroxidase type VI-A, fibrous DEAE-cellulose, benzamidine, iodoacetamide, phenyl methyl sulfonyl fluoride (PMSF), N-N′-1,2 phenylenedimaleimide (o-PDM), N-N′-1,4 phenyllenedimaleimide (p-PDM), gel filtration molecular weight protein marker kit, Staphylococcus aureus V8 protease, concanavalin A (Con A), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), methyl-α-d-mannopyranoside, methyl-α-d-glucopyranoside, Sigma; Q-Sepharose, S-Sepharose, Sefacryl S-300, Pharmacia; pre-stained high molecular weight protein markers, Gibco BRL; sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), nitrocellulose (0.45 μm pore size), Pierce; broad range isoelectric focusing calibration kit (3–10), Immobilin dry strips (pH 5–8), ampholites (pH 3–10), BioRad. All other chemicals were of the highest quality grade available.