CBSX2 Antibody

What is CBSX2 and what is its biological function?

CBSX2 is a CBS domain-containing protein localized in chloroplasts that functions as a regulator of redox systems. It contains a single pair of CBS domains and plays an essential role in modulating the activity of thioredoxin systems. Research shows that CBSX2 is required for the efficient oxidation of chloroplast redox-regulated enzymes, particularly during transitions from light to darkness. CBSX2 interacts with various thioredoxins (Trxs) including Trx f1, m1, m2, m4, y2, and CDSP32/Trx L1, as well as other redox-related proteins such as peroxiredoxins and NADPH-dependent thioredoxin reductase C (NTRC) . Unlike its homolog CBSX1 (which is also localized in chloroplasts), CBSX2 has been shown to specifically affect non-photochemical quenching (NPQ) induction during dark-light transitions .

What applications are CBSX2 antibodies typically used for?

Based on available research data, CBSX2 antibodies are primarily used for:

• Western blotting (WB) to detect CBSX2 protein expression levels

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) to study CBSX2 localization

• Co-immunoprecipitation (Co-IP) experiments to identify protein-protein interactions

• Examining redox regulation mechanisms in chloroplasts

• Studying the function of CBSX2 in mutant vs. wild-type plant models

For instance, researchers have successfully used tagged versions of CBSX2 (CBSX2-HA) in co-immunoprecipitation experiments to identify interaction partners, revealing connections to multiple chloroplast redox systems .

Which species are CBSX2 antibodies reactive with?

Commercial CBSX2 antibodies have been validated for reactivity with human and mouse CBSX2 proteins . When working with plant samples (particularly Arabidopsis), researchers should verify cross-reactivity or consider using epitope-tagged versions of CBSX2 with corresponding tag antibodies as demonstrated in previous research . Sequence alignment analysis between species can help predict potential cross-reactivity when selecting antibodies for non-validated species.

What is the difference between CBSX1 and CBSX2 proteins?

Despite both being localized in chloroplasts, CBSX1 and CBSX2 serve distinct functions:

• CBSX2 knockout mutants show altered NPQ induction during dark-light transitions, while CBSX1 mutants do not exhibit this phenotype

• The double mutant cbsx1cbsx2 displays identical defects to the single cbsx2 mutant, suggesting CBSX1 is not involved in NPQ induction and relaxation

• Phylogenetic analysis suggests that CBSX proteins localized in the same subcellular compartment are more closely related than those targeted to different compartments

This functional differentiation has important implications for designing experiments that target specific aspects of chloroplast redox regulation.

How can I optimize co-immunoprecipitation experiments using CBSX2 antibodies?

For successful co-immunoprecipitation (Co-IP) of CBSX2 and its interaction partners:

• Consider using epitope-tagged CBSX2 (e.g., CBSX2-HA) expressed in a cbsx2 mutant background to ensure specificity

• Use appropriate buffer conditions that preserve protein-protein interactions while minimizing non-specific binding

• Include adenylates (AMP, ATP) in your buffer system when studying interactions with thioredoxins, as these nucleotides can modulate CBSX2-thioredoxin interactions

• When analyzing results by mass spectrometry, focus on unique peptides rather than just coverage to ensure confident identification of interaction partners

A previous study successfully used the mMACS HA isolation kit (Miltenyi Biotec) for affinity purification of CBSX2-HA and its interacting proteins, followed by LC-MS/MS analysis to identify the protein interactome .

What methodological approaches can detect the redox state of CBSX2 target proteins?

To effectively study CBSX2's role in redox regulation:

• Alkylation-based redox mobility shift assays: Treat samples with alkylating agents (such as N-ethylmaleimide or iodoacetamide) to label free thiols, then separate reduced and oxidized forms by non-reducing SDS-PAGE

• Redox western blot analysis: Block free thiols with an alkylating agent, reduce disulfides with DTT, label newly exposed thiols with a different alkylating agent, then detect with specific antibodies

• Time-course experiments: Analyze samples collected during transitions between light and dark to capture dynamic redox changes in CBSX2 targets like CF1γ, FBPase, and SBPase

This approach has successfully demonstrated that oxidation of CF1γ, FBPase, and SBPase is less efficient during light-to-dark transitions in cbsx2 mutants compared to wild-type plants .

How can protein-protein interactions between CBSX2 and thioredoxins be validated?

Multiple complementary approaches should be used:

• Yeast two-hybrid (Y2H) analysis: Using truncated ORFs encoding mature proteins without target peptides. Specific primers (such as CBSX_intern and CBSX_stop) can be designed for this purpose

• Pull-down assays: Incubate purified 6His-CBSX2 with potential interaction partners (e.g., TRX m1) in appropriate buffer conditions, with or without adenylates (AMP, ATP) and reducing agents (DTE)

• Co-immunoprecipitation from plant tissue: As demonstrated in previous research, this can identify physiologically relevant interactions

• Bimolecular fluorescence complementation (BiFC): To visualize interactions in planta

The table below summarizes key interactors identified by co-immunoprecipitation with CBSX2-HA:

This data demonstrates the wide range of CBSX2 interactors in the chloroplast redox network .

Why might Western blots using CBSX2 antibodies show multiple bands?

Multiple bands in Western blots using CBSX2 antibodies could result from:

• Post-translational modifications of CBSX2 (such as phosphorylation or redox-dependent modifications)

• Alternative splicing of CBSX2 transcripts

• Proteolytic degradation during sample preparation

• Cross-reactivity with other CBS domain-containing proteins

To address this issue:

• Include appropriate controls (CBSX2 knockout/mutant samples)

• Optimize extraction conditions to minimize proteolysis (use fresh samples and protease inhibitors)

• Consider using epitope-tagged CBSX2 with tag-specific antibodies in recombinant systems

• Perform peptide competition assays to confirm specificity

How can I resolve inconsistent immunoprecipitation results with CBSX2?

Inconsistent co-immunoprecipitation results may occur due to:

• Dynamic nature of CBSX2 interactions that are influenced by redox state

• Adenylate (ATP/AMP) levels affecting CBSX2 binding to interaction partners

• Sample preparation conditions disrupting native protein complexes

For improved consistency:

• Standardize sample collection conditions (time of day, light conditions)

• Consider the redox state of the tissue, as CBSX2 functions in redox regulation

• Include adenylates in buffer systems when appropriate, as they can modulate CBSX2 interactions

• Perform crosslinking prior to extraction to stabilize transient interactions

• Use gentler extraction and wash conditions to preserve weak interactions

How should I interpret conflicting data regarding CBSX2 function in redox regulation?

Recent research has presented seemingly contradictory findings about CBSX2's role:

• Some studies suggest CBSX2 selectively inhibits the activities of m-type TRXs, with ATP reversing this effect

• Other research proposes that CBSX1 and CBSX2 may not function as Trx regulators for activation of Calvin-Benson cycle enzymes (FBPase and SBPase) in light conditions

• New findings indicate CBSX2 is involved in the oxidation of chloroplast redox-regulated proteins, including Calvin cycle enzymes and CF1γ

To reconcile these findings:

• Consider the specific experimental conditions (light vs. dark, in vitro vs. in vivo)

• Examine the precise redox targets being measured

• Use multiple complementary approaches to verify your findings

• Control for the presence of adenylates, which may modulate CBSX2 activity

• Consider CBSX2 may have distinct functions in different physiological contexts

How can CBSX2 antibodies be used to study dynamic changes in protein-protein interactions during light/dark transitions?

To investigate dynamic changes in CBSX2 interactions:

• Perform time-course experiments with samples collected at specific intervals during light/dark transitions

• Use rapid crosslinking to capture transient interactions at each timepoint

• Combine co-immunoprecipitation with quantitative proteomics (such as SILAC or TMT labeling) to measure changes in interaction stoichiometry

• Consider including phosphatase inhibitors to preserve potential phosphorylation-dependent interactions

• Compare results from wild-type plants versus various redox mutants (ntrc, 2cp)

This approach would build upon existing findings showing that oxidation of CF1γ, FBPase, and SBPase is less efficient during light-to-dark transitions in cbsx2 mutants .

What role does CBSX2 play in regulating NTRC activity and how can this be studied?

CBSX2 has been shown to interact with NTRC, suggesting it may regulate NTRC activity. Recent biochemical studies confirmed that CBSX protein acts as a negative regulator of NTRC in the presence of AMP, with even low concentrations of CBSX significantly inhibiting NTRC activity .

To study this regulatory mechanism:

• Use purified recombinant proteins to perform in vitro NTRC activity assays with varying concentrations of CBSX2 and adenylates

• Compare the redox status of known NTRC targets in wild-type, cbsx2 mutant, and NTRC overexpression plants

• Utilize Y2H experiments to map the specific interaction domains between CBSX2 and NTRC

• Develop FRET-based biosensors to monitor CBSX2-NTRC interactions in real-time in planta

• Create point mutations in CBSX2's adenylate-binding sites to test how nucleotide binding affects the regulation of NTRC

How do CBSX2-peroxiredoxin interactions affect reactive oxygen species (ROS) signaling?

Co-immunoprecipitation experiments have identified multiple peroxiredoxins (2CPA, 2CPB, PrxIIE, and PrxQ) as CBSX2 interactors . This suggests CBSX2 may play a role in ROS signaling and detoxification.

To investigate this:

• Compare H2O2 levels and peroxiredoxin oxidation states in wild-type versus cbsx2 mutant plants under various stress conditions

• Use fluorescent ROS sensors to monitor real-time changes in ROS levels in response to altered CBSX2 expression

• Perform double mutant analysis combining cbsx2 with mutations in various peroxiredoxins

• Investigate how adenylate levels affect CBSX2-peroxiredoxin interactions and subsequent ROS detoxification

• Develop in vitro reconstitution assays with purified components to determine if CBSX2 directly affects peroxiredoxin activity

What approaches can be used to study the adenylate sensing function of CBSX2?

CBSX2 contains CBS domains that may function as adenylate sensors. To investigate this:

• Design mutations in the putative adenylate-binding sites of CBSX2 and express these in the cbsx2 mutant background

• Use isothermal titration calorimetry (ITC) to measure binding affinities of CBSX2 for different adenylates

• Perform structural studies (X-ray crystallography or cryo-EM) of CBSX2 in the presence and absence of adenylates

• Develop FRET-based biosensors to monitor adenylate binding to CBSX2 in vivo

• Compare the interactomes of wild-type CBSX2 versus adenylate-binding mutants

This approach would help clarify how CBSX2 integrates metabolic signals (via adenylate sensing) with redox regulation in chloroplasts.

How could CBSX2 antibodies be used to investigate cross-talk between redox regulation and other signaling pathways?

CBSX2 antibodies could be valuable tools for studying the integration of redox signaling with other cellular pathways:

• Combine CBSX2 immunoprecipitation with phosphoproteomics to identify potential regulatory phosphorylation events

• Investigate changes in the CBSX2 interactome under different environmental stresses (drought, high light, temperature extremes)

• Compare CBSX2 protein complexes at different developmental stages

• Examine how hormonal signaling affects CBSX2 interactions and function

• Develop proximity labeling approaches (BioID or APEX) with CBSX2 as the bait to capture transient interactions in specific cellular conditions

This integrative approach would help elucidate how CBSX2 functions within the broader signaling network of plant cells.