Immunological detection of autophagy and mTORC1-related proteins
Harper JW, Sharan Sharan Swarup
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Abstract
Here we present a general protocol for immunological detection by Western blotting MTOR, MTOR (pS2448), ULK1, ULK1 (pS757), p70S6K, p70S6K (pT389), SQSTM1, CALCOCO2, MAP1LC3B, GABARAP, TFEB, TFE3, PGRN, HSP90, and PCNA
Steps
Western blotting
Lyse cell pellets by homogenization in KPBS buffer, urea buffer, or RIPA buffer with protease and phosphatase inhibitors. FOr some experiments, we employ 293 cells or alternatively HeLa cells with or without the GRN gene, created by CRISPR-based gene editing (DOI: dx.doi.org/10.17504/protocols.io.4r3l2oxqqv1y/v1).
Determined total protein concentration by BCA or Bradford assay. Normalize samples within a set of samples with additional buffer. Add NuPAGE LDS buffer (4X) plus NuPAGE reducing agent (10X).
Load samples onto 4-12% NuPAGE Bis-Tris gels (ThermoFisher), and separate by electrophoresis in MES buffer.
Transfer proteins to PVDF or nitrocellulose membranes by standard wet transfer in 20% methanol.
Stain membranes with REVERT 700 total protein stain following manufacturer’s instructions, and image total protein with a ChemiDoc MP (Bio-Rad) at 680 nm.
De-stain with REVERT reversal solution for 5 min. Block membranes with tris-buffered saline (TBS) (5% non-fat dry milk) at room temperature for 30-60 min.
Incubate membranes overnight at 4ºC with primary antibody solution in TBS with 0.1% Tween-20 (TBST). Wash six times with TBST for 5 min each. Incubate in secondary antibody solution in TBST (plus 0.01% SDS) for 1h at room temperature.
Wash membranes four times with TBST for 5 min each.
When using HRP-conjugated secondary antibodies (Bio-Rad or Cell Signaling Technology), apply luminol and hydrogen peroxide solution to membrane for 2 min, and image membrane with a ChemiDoc MP using the chemiluminescent setting.
When using Li-Cor fluorescent secondary antibodies, blot membranes dry and image with a ChemiDoc MP at either 800 nm or 680 nm, depending on the secondary antibody.
