Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

Abstract

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

Functional Characterization of a Novel Genetic Variant in Desmin (p.Glu353dup) Causing Myofibrillar Myopathy and Generation of Patient-Derived Induced Pluripotent Stem Cells for Disease Modeling

“…With this technique, the quaternary structures of the protein are maintained and differences in folding can be observed in a differential run on the polyacrylamide gel electrophoresis (PAGE). Protein samples were obtained from HEK cells that overexpressed DES_WT or DES_dupGGA for 72 h. Cell lysis, electrophoresis and Western blot were performed as previously described [57]. As control, a denaturing Western blot was also performed using SDS-PAGE.…”

Prediction of Metal Ion Binding Sites in Proteins from Amino Acid Sequences by Using Simplified Amino Acid Alphabets and Random Forest Model

“…In in - vitro condition, the unfolded polypeptide may are observed to interact with metal ions that direct the polypeptide folding process [ 2 ]. Identification of metal binding through experimental procedures like the use of metal ion affinity column chromatography [ 3 , 4 ], electrophoretic mobility shift assay [ 5 , 6 ], absorbance spectroscopy [ 7 ], gel electrophoresis [ 8 ], nuclear magnetic resonance spectroscopy [ 9 – 11 ], and mass spectrometry [ 3 , 12 ] require tedious steps and specific instruments, making them expensive and may be unsuitable for unknown targets. In this aspect, there is a need for computational predictors of protein binding metal ion in order to reduce time and cost.…”

Trauma diagnostic-related target proteins and their detection techniques

“…In contrast to denaturing methods, native gel electrophoresis does not employ charged denaturants such as SDS, allowing the preservation of the native protein structure. Consequently, the migration of proteins is determined by their inherent charge (Ref 135…”