MICAL-Oxidized (Pyrene labeled) Actin Protein (>95% pure): Rabbit Skeletal Muscle

MICAL-Oxidized Pyrene Actin Protein (>95% pure): Rabbit Skeletal Muscle (250 micrograms)

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.

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

Click here for a detailed method 

For more information contact:  signalseeker@cytoskeleton.com

Associated Products:

MOXtrue™ 6xHis MICAL-1 Protein (Cat. # MIC01)

MOXtrue™ 6xHis MsrB2 Protein (Cat. # MB201)

MOXtrue™ MICAL-oxidized Actin (Cat. # MXA95)

Rabbit Skeletal Muscle Actin (Cat. # AKL95)

Pyrene Labeled Rabbit Skeletal Muscle Actin (Cat.# AP05)

For product Datasheets and MSDSs please click on the PDF links below.

Certificate of Analysis:  Lot 001

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