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：

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.

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 μM).  2 μg 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 

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 μM) or 0.2 mg/ml (4.6 μM) (see method). Samples were then incubated with 2x polymerization buffer.  Upon actin polymerization fluorescence was detected with a spectrophotometer. A.U. = arbitrary units