For filter trap assays, native fractions were spotted onto nitrocellulose 0.2 µm membranes (Protran, GE) using a dot blot vacuum device (Whatman, Maidstone, UK). Nitrocellulose membranes were fixed for 30 min in PBS with PFA 0.4% (v/v) (Sigma-Aldrich, Saint-Louis, MO, USA) final concentration. After three washes with PBS, membranes were blocked with 5% (w/v) skimmed powder milk in PBS-Tween 0.5% (v/v) and probed with primary and secondary antibodies in PBS-Tween with 4% (w/v) BSA (Table 1 ). Immunoreactivity was whether visualized by chemiluminescence or infrared using Clarity ECL and Chemidoc (Biorad, Hercules, CA, USA) or Odissey systems (Li-Cor, Lincoln, NE, USA)) respectively.
Dot blot vacuum device
Manufactured by Cytiva
Sourced in United Kingdom
The Dot blot vacuum device is a lab equipment used for the rapid and efficient transfer of samples onto a membrane or filter. It applies a vacuum to facilitate the binding of samples, enabling efficient processing of multiple samples simultaneously.
Automatically generated - may contain errors
3 protocols using dot blot vacuum device
1
Nitrocellulose Membrane-Based Filter Trap Assay
2
Dot Blot Assay for Native Protein Detection
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For filter trap assays, native Sarkospin fractions were spotted onto layered nitrocellulose and PVDF 0.2 µm membranes (Protran, GE) using a dot blot vacuum device (Whatman). Membranes were fixed for 30 min in PBS with PFA 0.4% (v/v) (Sigma) final concentration. After three washes with PBS, membranes were blocked with 5% (w/v) skimmed powder milk in PBS-Tween 0.5% (v/v) and probed with primary and secondary antibodies in PBS-Tween with 4% (w/v) BSA (Antibody Table). Immunoreactivity was whether visualized by chemiluminescence or infrared using Clarity ECL and Chemidoc (Biorad) or Odissey systems (Li-Cor) respectively.
3
Filter Trap Assay for Protein Quantification
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For filter trap assays, native fractions were spotted onto nitrocellulose 0.2-μm membranes (Protran, GE) using a dot blot vacuum device (Whatman). Nitrocellulose membranes were fixed for 30 min in PBS with paraformaldehyde (PFA) at 0.4% (v/v) (Sigma-Aldrich) final concentration. After three washes with PBS, membranes were blocked with 5% (w/v) skimmed powder milk in PBS-Tween 0.5% (v/v) and probed with primary and secondary antibodies in PBS-Tween with 4% (w/v) bovine serum albumin (table S1). Immunoreactivity was visualized by chemiluminescence (Bio-Rad). The amount of the respective protein in each fraction was determined by the Image Studio Lite software after acquisition of chemiluminescent signals with a ChemiDoc digital imager (Bio-Rad). The profiles obtained were normalized and plotted with SD, all with respective Student’s t test and analysis of variance (ANOVA) using the Prism software.
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