Western Blot Analysis of ADAM10 Protein

Total protein from cells and tissues were extracted with RIPA buffer containing protease inhibitor cocktail and EDTA. Proteins were electrophoresed and transferred to membranes as described previously^{36 (link)}. The membranes were then blocked with 5% Blotting-Grade Blocker (Bio-Rad) in TBS-T (50 mM Tris, 138 mM NaCl, pH 7.4, 0.1% Tween-20) and subsequently incubated at 4 °C overnight with rabbit anti-ADAM10 polyclonal antibody (LifeSpan BioSciences,) or rabbit anti-α-tubulin polyclonal antibody (Cell Signaling Technology) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad).

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Ikeda A., Shahid S., Blumberg B.R., Suzuki M, & Bartlett J.D. (2019). ADAM10 is Expressed by Ameloblasts, Cleaves the RELT TNF Receptor Extracellular Domain and Facilitates Enamel Development. Scientific Reports, 9, 14086.

Publication 2019

Blotting-Grade Blocker rabbit anti-α-tubulin polyclonal antibody horseradish peroxidase (HRP)-conjugated secondary antibodies

Anti antibody Antibodies Buffer Cells Edta Horseradish peroxidase Membranes Nacl Protease inhibitor Proteins Rabbit Ripa Tissues Tris Tween 20 α tubulin

Corresponding Organization :

Other organizations : The Ohio State University, Augusta University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

ADAM10 protein levels
α-tubulin protein levels

control variables

RIPA buffer containing protease inhibitor cocktail and EDTA for protein extraction
Electrophoresis and transfer conditions as described in previous study (link provided)
Blocking with 5% Blotting-Grade Blocker in TBS-T
Incubation with rabbit anti-ADAM10 polyclonal antibody or rabbit anti-α-tubulin polyclonal antibody at 4°C overnight
Incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies

controls

Positive control: Unknown
Negative control: Unknown

Annotations

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