Recombinant Human MCAT Protein: Research Guide & Usage

Recombinant Human MCAT Protein is a key enzyme component of the mitochondrial fatty acid synthesis type II (mtFAS II) pathway, enabling researchers to investigate malonyl-CoA transfer, lipoic acid biosynthesis, and mitochondrial metabolic regulation in vitro.

Protein Name

Recombinant Human MCAT Protein

SKU

rp375199

Full Gene Name

Malonyl-CoA:ACP Transacylase (MCAT)

Biological Function

Transfers the malonyl group from malonyl-CoA to acyl carrier protein (ACP) in the mitochondrial fatty acid synthesis type II pathway

Research Relevance

Involved in lipoic acid biosynthesis and mitochondrial respiratory chain complex assembly

Recommended Storage

Store at -20°C or -80°C; avoid repeated freeze-thaw cycles

Biological Background and Function of MCAT

MCAT (Malonyl-CoA:ACP Transacylase, also known as Malonyl-CoA Transacylase, Mitochondrial) is a nuclear-encoded mitochondrial enzyme that plays an essential role in the mitochondrial fatty acid synthesis type II (mtFAS II) pathway. Unlike the cytoplasmic fatty acid synthesis type I system, the mtFAS II pathway is located in the mitochondrial matrix and shares mechanistic similarities with bacterial fatty acid synthesis.

The primary catalytic function of MCAT is the transfer of the malonyl group from malonyl-CoA to the phosphopantetheine arm of mitochondrial acyl carrier protein (ACP, encoded by NDUFAB1), generating malonyl-ACP. This transacylation reaction is a committed and rate-influencing step in elongation cycles of the mtFAS II pathway.

Beyond fatty acid elongation, the mtFAS II pathway and MCAT activity are functionally linked to the biosynthesis of lipoic acid (LA), an essential cofactor for several key mitochondrial enzyme complexes, including pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH). Consequently, MCAT is indirectly critical for oxidative phosphorylation and mitochondrial energy metabolism.

Protein Characteristics and Formulation

Recombinant Human MCAT Protein (SKU: rp375199) is produced using recombinant expression technology to ensure consistent quality and biological activity suitable for in vitro research applications. Researchers should consult the specific product datasheet for confirmed details on the following characteristics, as formulation may vary by lot:

• Expression System: Typically expressed in E. coli or a mammalian/insect cell system; refer to the datasheet for the confirmed host system for rp375199.
• Purity: Recombinant proteins of this class are generally supplied at >85–95% purity as assessed by SDS-PAGE; confirm the exact specification on the product datasheet.
• Tag Information: May include an affinity tag (e.g., His-tag) to facilitate purification; verify tag position and sequence on the datasheet.
• Formulation: Typically supplied in a phosphate-buffered saline (PBS) or Tris-based buffer, often with a stabilizer such as glycerol; exact buffer composition should be confirmed on the datasheet.
• Molecular Weight: Determined by the mature protein sequence plus any fusion tag; confirm expected band size on SDS-PAGE from the datasheet.

Always refer to the current Aladdin Scientific product datasheet for lot-specific certificate of analysis (CoA) data, including endotoxin levels, bioactivity assay results, and confirmed purity.

Handling, Storage, and Reconstitution Protocol

Proper handling and storage of Recombinant Human MCAT Protein are critical to maintaining enzymatic activity and experimental reproducibility. Follow the guidelines below as general best practices; always defer to lot-specific instructions on the product datasheet.

Storage Conditions

• Store lyophilized protein at -20°C or -80°C upon receipt.
• Liquid formulations should be stored at -80°C for long-term storage.
• Avoid repeated freeze-thaw cycles, as these can cause protein aggregation and loss of activity. Aliquot the protein into single-use volumes before freezing.

Reconstitution (if lyophilized)

1. Allow the vial to equilibrate to room temperature for 10–15 minutes before opening.
2. Reconstitute in sterile deionized water or the buffer recommended on the datasheet to the suggested stock concentration.
3. Gently swirl or pipette to dissolve; do not vortex vigorously.
4. Allow to stand for 5 minutes at room temperature, then centrifuge briefly if any particulates are visible.
5. Aliquot immediately and store as directed.

Working Dilutions

Prepare working dilutions in a compatible assay buffer immediately before use. Avoid prolonged incubation of diluted protein at room temperature. Carry an appropriate positive control in each experiment to verify protein activity.

Research Applications

Recombinant Human MCAT Protein is a versatile reagent suitable for a range of biochemical and cell biology research applications, particularly those focused on mitochondrial metabolism and fatty acid biology:

• Enzyme Activity Assays: MCAT can be used in in vitro transacylation assays to characterize its kinetic parameters (Km, Vmax) or to screen for small-molecule inhibitors and activators relevant to metabolic disease research.
• Protein-Protein Interaction Studies: As an integral component of the mtFAS II machinery, MCAT can be used in pull-down assays, co-immunoprecipitation experiments, or surface plasmon resonance (SPR) studies to map its interactions with ACP (NDUFAB1) and other pathway components.
• Structural Biology: Recombinant MCAT is suitable as a substrate for X-ray crystallography or cryo-electron microscopy studies to elucidate the structural basis of malonyl transfer.
• Antibody Validation: Recombinant MCAT protein serves as a positive control antigen for validating anti-MCAT antibodies in Western blot, ELISA, and immunoprecipitation applications.
• Metabolic Disease Research: Given the link between mtFAS II, lipoic acid synthesis, and mitochondrial dysfunction, MCAT is a relevant target in research on metabolic syndromes, neurodegeneration, and mitochondrial diseases.
• Drug Discovery: MCAT's conserved active site and essential role in mitochondrial function make it a potential target for mechanism-of-action studies in drug discovery pipelines.

Quality Control and Troubleshooting

To ensure reliable experimental outcomes when working with Recombinant Human MCAT Protein, consider the following quality control measures and troubleshooting guidance:

Quality Control Checks

• Verify protein identity and purity by SDS-PAGE upon receipt, comparing the observed band size with the expected molecular weight stated on the datasheet.
• For activity-dependent applications, run a functional activity assay using validated conditions before integrating the protein into novel experimental workflows.
• Check endotoxin levels (reported on the CoA) if the protein will be used in cell-based assays, as endotoxin contamination can confound results.

Common Issues and Solutions

• Loss of Activity: Most commonly caused by repeated freeze-thaw cycles or improper storage temperature. Ensure protein is aliquoted into single-use volumes and stored at the recommended temperature.
• Protein Aggregation: May occur upon thawing or during prolonged incubation. Briefly centrifuge (e.g., 10,000 × g, 5 min, 4°C) before use to remove aggregates.
• Unexpected Band Size on SDS-PAGE: Account for the affinity tag and any signal peptide cleavage. Compare with the datasheet; minor deviations may reflect glycosylation or post-translational modifications depending on the expression system.
• Low Assay Signal: Confirm that the assay buffer, pH, and cofactor concentrations (e.g., CoA, ACP substrate) are appropriate for MCAT activity. Consult published literature for established assay conditions.

For product-specific technical support, contact Aladdin Scientific's technical support team, referencing SKU rp375199 and your lot number.

Frequently asked questions

What is the biological function of the human MCAT protein?

MCAT (Malonyl-CoA:ACP Transacylase) catalyzes the transfer of a malonyl group from malonyl-CoA to mitochondrial acyl carrier protein (ACP), a critical step in the mitochondrial type II fatty acid synthesis (mtFAS II) pathway. This pathway is essential for the biosynthesis of lipoic acid, an indispensable cofactor for key mitochondrial enzyme complexes involved in energy metabolism.

How should Recombinant Human MCAT Protein (rp375199) be stored to maintain activity?

Store the protein at -20°C or -80°C as indicated on the datasheet. To prevent loss of activity due to protein degradation or aggregation, avoid repeated freeze-thaw cycles by aliquoting the protein into single-use volumes immediately upon first thaw. Always refer to the lot-specific datasheet for the most accurate storage recommendations.

What research applications is Recombinant Human MCAT Protein suitable for?

Recombinant Human MCAT Protein is suitable for enzyme kinetics and inhibitor screening assays, protein-protein interaction studies (e.g., pull-down, SPR), structural biology investigations, antibody validation (Western blot, ELISA), and research into mitochondrial metabolic diseases, lipoic acid synthesis, and drug discovery targeting mitochondrial fatty acid metabolism.

How can I confirm the purity and identity of the received MCAT protein?

Upon receipt, run an SDS-PAGE analysis and compare the observed band with the expected molecular weight listed on the product datasheet and certificate of analysis (CoA). For functional confirmation, perform an in vitro transacylation activity assay under established conditions. If endotoxin-free performance is required for cell-based assays, verify the endotoxin level reported on the CoA.

Can Recombinant Human MCAT Protein be used in cell-based assays?

Recombinant Human MCAT Protein is primarily designed for in vitro biochemical applications. If you plan to use it in cell-based contexts, verify that the endotoxin level on the CoA is acceptable for your cell type, and confirm that the buffer formulation is compatible with your assay system. Consult the datasheet and contact Aladdin Scientific technical support for guidance specific to your application.

References

1. Hiltunen JK, Schonauer MS, Autio KJ, Mittelmeier TM, Kastaniotis AJ, Dieckmann CL. Mitochondrial fatty acid synthesis type II: more than just fatty acids. J Biol Chem. 2009;284(14):9011-9015. doi:10.1074/jbc.R800074200
2. Kastaniotis AJ, Autio KJ, Keratar JM, et al. Mitochondrial fatty acid synthesis, fatty acids and mitochondrial physiology. Biochim Biophys Acta Mol Cell Biol Lipids. 2017;1862(1):39-48. doi:10.1016/j.bbalip.2016.08.011
3. Autio KJ, Kastaniotis AJ, Pospiech H, et al. An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing. FASEB J. 2008;22(2):569-578. doi:10.1096/fj.07-8986com

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.