MICAL interacts with F-actin and uses NADPH as a cofactor to oxidize actin at Met44 and Met47 (b-actin nomenclature). Functionally, oxidation of Met44 has a profound effect on actin polymerization because the residue resides in the D-loop of subdomain 2 of the protein, which is critical for actin subunit contacts; thus, upon oxidation, Met44 becomes negatively charged and interferes with actin monomer-monomer interaction and promotes F-actin severing and depolymerization. Regulation of actin oxidation at Met44/Met47 has been shown to destabilize F-actin in vivo and to play a key role in a growing number of cellular processes. As part of the MOXtrue™ product line, Pyrene labeled rabbit skeletal muscle actin protein (MICAL-oxidized) (MXAP95) has been enzymatically oxidized at methionines 44 and 47 with the MICAL flavoprotein monoxygenase protein. Purified MICAL-oxidized (pyrene labeled) actin has reduced susceptibility to subtilisin A cleavage at M47/G48 by > 90%, and has also been validated for downstream applications such as actin polymerization assays.
To learn more about using MICAL-oxidized Actin as a research tool see our datasheet
To learn more about the MICAL/MsrB/Actin physiological redox system see our Newsletter
Each lot of purified protein is quality controlled to provide high batch to batch consistency, see COA documents.
Validated Applications
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Pyrene labeled Actin Protein (MICAL-Oxidized) Purity Determination
A 50 μg sample of pyrene labeled actin protein (MICAL-oxidized) was separated by electrophoresis in a 4- 20% tris-glycine gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision RedTM Protein Assay Reagent (Cat. # ADV02). Mark12 standard molecular weight markers are from Invitrogen.
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Subtilisin Assay on MICAL-Oxidized Pyrene Actin vs Native Pyrene Actin
Pyrene labeled actin (Cat. # AP05) and MICAL-oxidized (pyrene labeled) actin (Cat. # MXAP95) was diluted to 0.1 mg/ml (2.3 mM). 2 mg of each sample was then left untreated, or treated with subtilisin (1:200 w/w) for 15 min. Samples were then separated by SDS-PAGE and visualized with Coomassie staining.
Click here for a detailed method
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Actin Polymerization of Oxidized Pyrene Actin Versus Native Pyrene Actin
Pyrene-labeled actin (Cat # AP05) and MICAL-oxidized (pyrene labeled) actin (Cat. # MXAP95) were diluted to 0.1 mg/ml (2.3 mM) or 0.2 mg/ml (see method). Samples were then incubated with 2x polymerization buffer. Upon actin polymerization fluorescence was detected with a spectrophotometer. A.U. = arbitrary units
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