Recombinant Human Aconitase 2 Protein: Protocol & Usage Guide
Recombinant Human Aconitase 2 Protein: Protocol & Usage Guide
Recombinant Human Aconitase 2 Protein is a mitochondrial matrix enzyme that catalyzes the reversible isomerization of citrate to isocitrate in the TCA cycle and serves as a critical research tool for studying iron-sulfur cluster biology, oxidative stress responses, and mitochondrial energy metabolism.
- Protein Name
- Recombinant Human Aconitase 2 (ACO2)
- SKU
- rp367182
- Biological Function
- Catalyzes reversible isomerization of citrate to isocitrate in the mitochondrial TCA cycle
- Cofactor
- Iron-sulfur [4Fe-4S] cluster required for enzymatic activity
- Host Expression System
- Recombinant expression system (refer to product datasheet for confirmed host)
- Recommended Storage
- -80°C for long-term storage; avoid repeated freeze-thaw cycles
Overview of Recombinant Human Aconitase 2 Protein
Aconitase 2 (ACO2), also known as mitochondrial aconitase, is a key enzyme of the tricarboxylic acid (TCA) cycle located in the mitochondrial matrix. It catalyzes the stereospecific isomerization of citrate to isocitrate via a cis-aconitate intermediate, a reaction essential for cellular energy production.
ACO2 contains a catalytic iron-sulfur ([4Fe-4S]) cluster that is sensitive to reactive oxygen species (ROS), making the enzyme a functional sensor of mitochondrial oxidative stress. Loss of ACO2 activity has been linked to neurodegenerative diseases, cancer metabolic reprogramming, and mitochondrial dysfunction.
Recombinant Human Aconitase 2 Protein (SKU: rp367182) from Aladdin Scientific is intended for use in biochemical assays, enzyme kinetics studies, and as a positive control in western blot or activity assay workflows. Researchers studying the TCA cycle, iron-sulfur cluster assembly, or mitochondrial redox biology will find this reagent especially valuable.
Reconstitution and Handling Protocol
Proper reconstitution and handling of Recombinant Human Aconitase 2 Protein are critical for maintaining enzymatic activity. Follow these steps carefully:
- Centrifugation: Prior to opening, briefly centrifuge the vial (e.g., 5–10 seconds at low speed) to collect the lyophilized material at the bottom of the tube.
- Reconstitution Buffer: Reconstitute the protein in sterile deionized water or the buffer recommended on the product datasheet (typically a phosphate-buffered saline, pH 7.4, containing a carrier protein such as 0.1% BSA to stabilize activity). Refer to the specific datasheet for the recommended volume.
- Gentle Mixing: Do not vortex. Mix gently by pipetting up and down or by slow rotation to avoid denaturation.
- Incubation: Allow the reconstituted protein to equilibrate at room temperature for 5–10 minutes before use.
- Aliquoting: Prepare single-use aliquots immediately after reconstitution to minimize freeze-thaw cycles, which can reduce enzymatic activity.
Note: The [4Fe-4S] cluster of ACO2 is sensitive to oxidation. Where possible, handle the protein under low-oxygen conditions or on ice in a cold room to preserve activity.
Storage Conditions and Stability
To ensure optimal stability and shelf life of Recombinant Human Aconitase 2 Protein, adhere to the following storage guidelines:
- Unopened/Lyophilized: Store at -20°C or -80°C as indicated on the product datasheet. Keep away from frost-free freezers that undergo temperature cycling.
- Reconstituted Protein: Store working aliquots at -80°C. Short-term storage (up to 1 week) at 4°C is generally acceptable if a carrier protein (e.g., BSA) has been added.
- Freeze-Thaw Cycles: Limit to a maximum of 2–3 cycles. Each freeze-thaw cycle may result in progressive loss of enzymatic activity due to oxidation and aggregation of the iron-sulfur cluster.
- Expiry: Refer to the product label for the lot-specific expiration date. Do not use beyond the stated expiry.
Always record the date of reconstitution on the aliquot tubes. Discard any aliquots showing visible precipitation or turbidity.
Recommended Assay Conditions and Applications
Recombinant Human Aconitase 2 Protein is suitable for a range of biochemical and cell-free applications:
Aconitase Activity Assay
Aconitase activity is typically measured spectrophotometrically by monitoring the conversion of isocitrate to cis-aconitate at 240 nm, or by coupling the reaction to NADP+ reduction via isocitrate dehydrogenase (monitored at 340 nm). A standard assay buffer contains 50 mM Tris-HCl (pH 7.4), 0.6 mM MnCl₂, and substrate (isocitrate or citrate). Confirm exact assay conditions from a validated reference or the product insert.
Western Blot Positive Control
The recombinant protein can be used as a positive control to verify antibody specificity in western blot experiments targeting ACO2. Load an appropriate amount of recombinant protein alongside cell or tissue lysates for size confirmation (~82 kDa for human ACO2).
Iron-Sulfur Cluster Studies
ACO2 is widely used as a model protein in studies of [4Fe-4S] cluster assembly, disassembly, and oxidative inactivation. The recombinant form enables controlled in vitro experiments without the complexity of whole-cell systems.
Protein-Protein Interaction Studies
Recombinant ACO2 can be used in pull-down assays or co-immunoprecipitation setups to identify binding partners involved in mitochondrial metabolon assembly.
Quality Control and Troubleshooting
Maintaining assay quality when working with Recombinant Human Aconitase 2 Protein requires attention to several factors:
Quality Control Checks
- Confirm protein identity and purity by SDS-PAGE and/or western blot prior to use in critical experiments.
- Include a no-enzyme blank in all activity assays to account for non-enzymatic substrate conversion.
- Use fresh substrate solutions to avoid background absorbance artifacts.
Common Issues and Solutions
| Issue | Likely Cause | Recommended Action |
|---|---|---|
| Low or absent enzyme activity | Oxidation of [4Fe-4S] cluster; repeated freeze-thaw; improper reconstitution | Use fresh aliquot; reconstitute under low-oxygen conditions; add reducing agent (e.g., DTT at 1–5 mM) if compatible with downstream assay |
| Protein precipitation | Freeze-thaw damage; incorrect buffer conditions | Centrifuge at 10,000 × g for 10 min at 4°C and use supernatant; ensure correct pH and ionic strength |
| Non-specific bands on western blot | Antibody cross-reactivity; sample overloading | Optimize antibody dilution; reduce sample load |
Frequently asked questions
What is the recommended reconstitution buffer for Recombinant Human Aconitase 2 Protein?
Recombinant Human Aconitase 2 Protein is generally reconstituted in sterile deionized water or phosphate-buffered saline (pH 7.4) supplemented with a carrier protein such as 0.1% BSA to enhance stability. Always refer to the specific product datasheet (SKU: rp367182) for the exact recommended buffer and volume.
How should I store the recombinant protein to preserve aconitase enzymatic activity?
Store lyophilized protein at -20°C or -80°C as directed on the product label. After reconstitution, aliquot immediately and store at -80°C. Limit freeze-thaw cycles to no more than 2–3, as the [4Fe-4S] catalytic cluster is susceptible to oxidative inactivation with each cycle. Short-term storage at 4°C (up to one week) is acceptable if BSA is present.
Can Recombinant Human Aconitase 2 Protein be used as a positive control in western blot experiments?
Yes, Recombinant Human Aconitase 2 Protein (rp367182) is suitable as a positive control for western blot experiments using ACO2-targeting antibodies. Human ACO2 has an approximate molecular weight of 82 kDa. Load an appropriate quantity alongside tissue or cell lysates to confirm correct band size and antibody specificity.
Why might I observe low or absent aconitase activity in my assay?
Low activity most commonly results from oxidation of the oxygen-sensitive [4Fe-4S] cluster during handling, multiple freeze-thaw cycles, or improper reconstitution. To mitigate this, handle the protein on ice or under reduced-oxygen conditions, use a freshly prepared aliquot, and consider including a mild reducing agent such as 1–5 mM DTT in the assay buffer if compatible with your downstream application.
What downstream applications is this recombinant protein validated for?
Recombinant Human Aconitase 2 Protein is commonly used in enzymatic activity assays (spectrophotometric), western blot positive controls, iron-sulfur cluster assembly and oxidation studies, and protein-protein interaction assays such as pull-downs. Please consult the product datasheet for the specific validated applications and performance data for lot rp367182.
References
- Robbins AH, Stout CD. The structure of aconitase. Proteins. 1989;5(4):289–312.
- Gardner PR. Aconitase: sensitive target and measure of superoxide. Methods Enzymol. 2002;349:9–23.
- Beinert H, Kennedy MC, Stout CD. Aconitase as iron-sulfur protein, enzyme, and iron-regulatory protein. Chem Rev. 1996;96(7):2335–2374.
Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.
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