The purified proteins were analyzed by gel filtration chromatography on a Superdex-200 analytical gel filtration column (GE HealthCare) equilibrated with PBS (pH 7.4) to determine their oligomeric state under native conditions. The column was calibrated using a broad range of molecular weight markers as indicated (GE HealthCare).
Molecular weight markers
Manufactured by GE Healthcare
Molecular weight markers are laboratory tools used to determine the molecular weight of proteins or other macromolecules. They are a set of pre-stained proteins or dyes with known molecular weights that are used as reference standards in electrophoresis techniques such as SDS-PAGE. Molecular weight markers help in the accurate estimation of the molecular weights of unknown protein samples by comparing their migration patterns to the known standards.
Automatically generated - may contain errors
3 protocols using molecular weight markers
1
Protein Oligomerization Analysis
2
Western Blot Analysis of Autophagy Markers
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Proteins were resolved on SDS–PAGE and electroblotted onto PVDF membranes. Membranes with transferred proteins were incubated with the primary monoclonal antibody and successively with the specific peroxidase conjugated secondary antibody. Monoclonal antibodies against Ub and p27 were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). SQSTM1/p62 (sequestosome 1, herein p62) mouse monoclonal antibody was from Sigma‐Aldrich S.r.L. (Milano, Italy) and the anti‐LC3B antibody was purchased from Cell Signaling Technology, Inc. The immunoblot detection was performed with ECL Western blotting detection reagents using a ChemiDoc MP system. Each gel was loaded with molecular weight markers in the range of 12 to 225 kDa (GE Healthcare). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was utilized as a control for equal protein loading: membranes were stripped and re‐probed with anti‐GAPDH monoclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany). Stripping buffer contained 200 mM glycine, 0.1% SDS, and 1% Tween 20. Immunoblot images were quantified using ImageJ 1.52p software (NIH, USA).
3
Size-Exclusion Chromatography of Protein Complexes
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Size-exclusion chromatography (SEC) experiments were carried out on an AKTA FPLC systems (GE Healthcare) using a Superdex 200 16/60 column (GE Heathcare) that was calibrated using molecular weight markers (GE Healthcare). The protein was clarified by centrifugation at maximum speed in a bench top centrifuge at 4°C, before loading onto the column. Protein and protein complexes were eluted using isocratic elution in 20 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol (v/v), 1 mM DTT at 0.5 ml/min.
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