Protein Fractionation and Immunoblotting Analysis

The obtained protein fractions were analyzed using standard SDS-polyacrylamide gel electrophoresis (PAGE), followed by Coomassie Brilliant Blue R-250 (Sigma-Aldrich) staining or Western blot. We used All Blue (Bio-Rad Laboratories) and Dual Xtra (Bio-Rad Laboratories) as molecular weight markers. Separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore), which was probed with polyclonal anti-human C-terminal AMCase27 (link), anti-mouse N-terminal AMCase, anti- mouse C-terminal AMCase27 (link), polyclonal goat anti-pepsin C (I-19) antibody (Santa Cruz) or monoclonal anti-β-actin (clone AC-15) (Sigma-Aldrich), followed by peroxidase-conjugated AffiniPure F (ab’)₂ Fragment Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories), AffiniPure Donkey Anti-Goat IgG-HRP (Jackson ImmunoResearch laboratories) or AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch laboratories). Bound antibodies were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The immunoblots were analyzed and quantified using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare) according to the manufacturer’s instructions.

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Ohno M., Kimura M., Miyazaki H., Okawa K., Onuki R., Nemoto C., Tabata E., Wakita S., Kashimura A., Sakaguchi M., Sugahara Y., Nukina N., Bauer P.O, & Oyama F. (2016). Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system. Scientific Reports, 6, 37756.

Publication 2016

Coomassie Brilliant Blue R-250 Immobilon-P monoclonal anti-β-actin (clone AC-15) AffiniPure F (ab’)2 Fragment Donkey Anti-Rabbit IgG (H+L) AffiniPure Donkey Anti-Goat IgG-HRP AffiniPure Donkey Anti-Mouse IgG (H+L) Immobilon Western Chemiluminescent HRP Substrate Luminescent Image Analyzer (ImageQuant LAS 4000,

Actin Anti igg Antibodies Antibody Clone Coomassie brilliant blue r Donkey Goat Human Immobilon Immobilon p Immunoblots Luminescent Membrane Molecular markers Mouse Mouse amcase Pepsin c Peroxidase Polyvinylidene fluoride Proteins Rabbit Sds polyacrylamide gel electrophoresis Western blot

Corresponding Organization :

Other organizations : Kogakuin University, Doshisha University, Juntendo University, RIKEN Center for Brain Science, Mayo Clinic in Florida

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Variable analysis

independent variables

Molecular weight markers used: All Blue (Bio-Rad Laboratories) and Dual Xtra (Bio-Rad Laboratories)

dependent variables

Separated proteins analyzed using SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot
Proteins detected using Coomassie Brilliant Blue R-250 (Sigma-Aldrich) staining or Western blot
Proteins transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore) and probed with various antibodies
Bound antibodies detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore)
Immunoblots analyzed and quantified using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare)

control variables

SDS-polyacrylamide gel electrophoresis (PAGE) used as a standard technique
Coomassie Brilliant Blue R-250 (Sigma-Aldrich) used as a standard staining method
Western blot used as a standard technique
Polyclonal anti-human C-terminal AMCase27, anti-mouse N-terminal AMCase, anti-mouse C-terminal AMCase27, polyclonal goat anti-pepsin C (I-19) antibody (Santa Cruz), and monoclonal anti-β-actin (clone AC-15) (Sigma-Aldrich) used as primary antibodies
Peroxidase-conjugated AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories), AffiniPure Donkey Anti-Goat IgG-HRP (Jackson ImmunoResearch laboratories), and AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch laboratories) used as secondary antibodies

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