Protein extracts of cells collected by centrifugation, washed twice in PBS, lysed in 1× lysis buffer (50 nM Tris–HCl, 70 mM 2‐mercaptoethanol, and 1% SDS) and the concentration of total protein was measured by the Bradford assay. Lysates were then boiled for 5 minutes, and subjected to 12% polyacrylamide SDS gels or 4%–12% SDS resolving gels (Invitogen). After electrophoresis, proteins were transferred to a nitrocellulose membrane using a submerged transfer apparatus (BioRad), filled with 25 mM Tris Base, 200 mM glycine, and 20% methanol. After blocking with 5% non‐fat dried milk in TBS‐T (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, and 0.1% Tween 20) the membrane was incubated with the primary antibodies diluted in TBS‐T and washed extensively. For the list of primary antibodies used see Supporting Information Figure S10 . The membrane was washed 3 times with TBS‐T and then incubated with the appropriate horseradish peroxidase linked secondary antibody (Amersham, Spain). The detection was performed with the Western Breeze Immunodetection Kit (Invitrogen, Spain).
Submerged transfer apparatus
Manufactured by Bio-Rad
The Submerged transfer apparatus is a laboratory equipment designed for the transfer of proteins or nucleic acids from polyacrylamide or agarose gels to a membrane. The apparatus facilitates the transfer process by immersing the gel and membrane in a buffer solution, allowing for efficient and controlled transfer of the target biomolecules.
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3 protocols using submerged transfer apparatus
1
Protein Extraction and Western Blot Analysis
2
Hippocampal Protein Expression Analysis by Western Blot
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Western blotting was performed as previously described [36 (link)]. Briefly, mouse hippocampal samples were homogenized in ice-cold IGEPAL RIPA buffer with protease inhibitors at 4 °C. 20 or 40 µg of total protein in Laemmli loading buffer was denatured at 85 °C for 10 min and separated by 7.5-, 10-, or 12% Criterion TGX Stain-free SDS-PAGE (Bio-Rad) and blotted onto 0.45 µm nitrocellulose membrane (Bio-Rad) by submerged transfer apparatus (Bio-Rad). Membranes were then blocked and incubated overnight with gentle agitation at 4 °C with the primary antibody (Additional File 1 : Table S1). Protein was detected by chemiluminescence (Clarity, Bio-Rad) according to the manufacturer’s instructions and signals were captured on a ChemiDoc Touch MP Imaging System (Bio-Rad).
3
Western Blotting Protocol for Mouse Hippocampal Samples
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Western blotting was performed as previously described [35] (link). Briefly, mouse hippocampal samples were homogenized in ice-cold IGEPAL RIPA buffer with protease inhibitors at 4 °C. 20 or 40 µg of total protein in Laemmli loading buffer was denatured at 85 °C for 10 min and separated by 7.5-, 10-, or 12% Criterion TGX Stain-free SDS-PAGE (Bio-Rad) and blotted onto 0.45 µm nitrocellulose membrane (Bio-Rad) by submerged transfer apparatus (Bio-Rad). Membranes were then blocked and incubated overnight with gentle agitation at 4 °C with the primary antibody (Supplementary Table 1, online resource 1). Protein was detected by chemiluminescence (Clarity, Bio-Rad) according to the manufacturer's instructions and signals were captured on a ChemiDoc Touch MP Imaging System (Bio-Rad).
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