SBT6.1 Antibody

What is SBT6.1 and why is it significant in plant research?

SBT6.1 (Subtilase subfamily 6 member 1) is a subtilisin-like protease in Arabidopsis thaliana that functions as a Site-1 protease (AtS1P). It belongs to a large family of proteases that includes 56 members in Arabidopsis . What makes SBT6.1 particularly significant is its evolutionary conservation - it is one of only two Arabidopsis SBTs that originated before the divergence of Metazoa and Viridiplantae, demonstrating functional conservation between animals and plants . Unlike most plant subtilases that are soluble and targeted to the cell wall, SBT6.1 is a membrane protein anchored by a C-terminal membrane-spanning helix to the Golgi and possibly the plasma membrane . This unique localization pattern allows it to participate in protein processing within the secretory pathway, making it a critical component in various signaling cascades, particularly in peptide hormone maturation and cell elongation processes.

What applications have SBT6.1 antibodies been validated for?

SBT6.1 polyclonal antibodies raised against Arabidopsis thaliana have been validated for several key applications in molecular biology research:

The antibody has been specifically generated against recombinant Arabidopsis thaliana SBT6.1 protein (amino acids 175-473) . When designing experiments, researchers should be aware that the specificity and sensitivity may vary depending on sample preparation and experimental conditions.

How should SBT6.1 antibodies be stored and handled?

For optimal performance and longevity of SBT6.1 antibodies, proper storage and handling are essential:

• Store at -20°C for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles that can degrade antibody quality

• Thaw on ice and centrifuge briefly before use to recover all material

• When working with the antibody, maintain cold chain to prevent degradation

• Follow manufacturer guidelines for specific buffer formulations that maintain stability

Improper storage can lead to antibody degradation, resulting in reduced sensitivity and increased background in experimental applications. For critical experiments, researchers should validate antibody performance with positive controls before proceeding with valuable samples.

How can I optimize Western blot protocols for SBT6.1 detection?

Optimizing Western blot protocols for SBT6.1 detection requires attention to several key factors:

• Sample preparation: Since SBT6.1 is a membrane-anchored protein localized to the Golgi and potentially plasma membrane , use extraction buffers containing mild detergents (0.5-1% Triton X-100 or NP-40) to efficiently solubilize the protein. Include protease inhibitors to prevent degradation during extraction.

• Gel selection: Use 10-12% SDS-PAGE gels for optimal separation of SBT6.1, which has a molecular weight of approximately 80-85 kDa.

• Transfer conditions: For membrane-bound proteins like SBT6.1, extend transfer time or use semi-dry transfer systems with methanol-containing buffers to improve transfer efficiency.

• Blocking optimization: Test both BSA and milk-based blocking solutions, as membrane proteins can sometimes show background with milk proteins. A 5% BSA solution in TBST often provides optimal results.

• Antibody dilution: Begin with a 1:1000 dilution for primary antibody incubation, adjusting based on signal strength. Overnight incubation at 4°C typically yields the best results for SBT6.1 detection.

• Controls: Always include a positive control (e.g., Arabidopsis tissue extract) and consider using SBT6.1 knockout/knockdown samples as negative controls to verify specificity.

For challenging samples, consider enriching membrane fractions through ultracentrifugation before loading to enhance detection sensitivity.

What approaches can be used to study SBT6.1-Serpin1 interactions?

Research has established that SBT6.1 proteolytic activity is regulated by the Serpin1 inhibitor, suggesting a complex regulatory network controlling cell elongation in Arabidopsis . Several methodological approaches can be employed to study this interaction:

• Tandem Affinity Purification (TAP): This has been successfully used to determine in vivo interaction between SBT6.1 and Serpin1. The TAP tag consisted of two IgG-binding domains of Staphylococcus aureus protein A (ZZ) and a calmodulin-binding peptide, separated by a tobacco etch virus protease cleavage site . This approach allows for stringent purification conditions to identify genuine interacting partners.

• Co-immunoprecipitation with SBT6.1 antibodies: Pull-down assays using SBT6.1 antibodies can help confirm the interaction with Serpin1 in different experimental conditions or genetic backgrounds.

• Bimolecular Fluorescence Complementation (BiFC): This can visualize the subcellular localization of the SBT6.1-Serpin1 interaction in living cells.

• In vitro inhibition assays: Examining how purified Serpin1 affects the proteolytic activity of SBT6.1 on known substrates can provide biochemical evidence of the regulatory relationship.

When interpreting results, researchers should consider that interactions may be transient or context-dependent. Affinity purification should incorporate steps to avoid common contaminants that could lead to false positives, as described in previous studies where systematic subtraction of experimental background was performed .

How can I use SBT6.1 antibodies to investigate subcellular localization?

Investigating the subcellular localization of SBT6.1 is critical for understanding its function in the secretory pathway. Several techniques can be employed using SBT6.1 antibodies:

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1-0.5% Triton X-100

  • Block with 3-5% BSA

  • Incubate with SBT6.1 primary antibody (1:100-1:500 dilution)

  • Use fluorescently-labeled secondary antibodies

  • Co-stain with organelle markers like ST-mCherry for Golgi

• Subcellular fractionation and immunoblotting:

  • Separate cellular compartments using differential centrifugation

  • Prepare fractions enriched for ER, Golgi, plasma membrane, and other compartments

  • Run Western blots with SBT6.1 antibody on each fraction

  • Use compartment-specific markers as controls for fractionation quality

• Immunogold electron microscopy:

  • For highest resolution localization studies

  • Requires specialized sample preparation and ultra-thin sectioning

  • SBT6.1 antibody is detected with gold-conjugated secondary antibodies

  • Allows precise localization within Golgi stacks and other membrane structures

Previous studies have found SBT6.1 primarily in the Golgi apparatus with possible plasma membrane localization . When conducting these experiments, it's important to include proper controls, such as testing the antibody in SBT6.1 knockout lines to confirm specificity, and using established subcellular markers to validate compartment identity.

How can SBT6.1 antibodies be used to study GOLVEN/CLEL peptide processing?

GOLVEN/CLEL peptides are signaling molecules that control cell elongation in Arabidopsis, and SBT6.1 plays a crucial role in their maturation . Using SBT6.1 antibodies can provide insights into this processing mechanism:

• In vitro processing assays:

  • Express and purify recombinant CLEL precursors

  • Incubate with immunoprecipitated SBT6.1 (using SBT6.1 antibodies)

  • Analyze cleavage products by mass spectrometry

  • Compare processing efficiency under different conditions

• Monitoring processing in cellular systems:

  • Design reporters with fluorescent tags flanking CLEL processing sites

  • Transfect into wild-type and SBT6.1 knockdown/knockout cells

  • Use SBT6.1 antibodies to correlate enzyme levels with processing efficiency

  • Analyze processing products by Western blot or mass spectrometry

Research has shown that SBT6.1 performs initial processing of CLEL6 in the Golgi, but additional processing at the N-terminus is required for complete maturation and activation . This suggests a sequential processing pathway involving multiple proteases. When designing experiments, researchers should consider that SBT6.1 cleavage sites are located considerably upstream of the mature peptide, indicating that it performs a pre-processing role rather than producing the final active peptide directly .

What methods can be used to study SBT6.1 activity in different cellular compartments?

Understanding the compartment-specific activity of SBT6.1 is crucial due to its localization in the secretory pathway. Several approaches can be employed:

• Compartment-targeted reporter substrates:

  • Design fluorogenic or chromogenic substrates containing SBT6.1 cleavage sites

  • Add targeting sequences to direct these reporters to specific compartments (ER, Golgi, plasma membrane)

  • Monitor cleavage in living cells or cellular fractions

• Inducible expression systems with compartment retention:

  • Generate constructs expressing SBT6.1 substrates with different retention signals:

    • KDEL for ER retention

    • Transmembrane domains for Golgi retention (such as the XylT construct used in prior studies)

    • Plasma membrane targeting sequences

  • Induce expression and monitor processing using SBT6.1 antibodies to detect enzyme levels

• Correlative microscopy approach:

  • Use fluorescently tagged substrates to visualize processing in real-time

  • Fix cells at different timepoints

  • Perform immunofluorescence with SBT6.1 antibodies

  • Correlate enzyme localization with substrate processing

Previous research has employed deletion constructs of CLEL6 precursors with different localization signals (secreted version, ER-retained with KDEL, and Golgi-anchored with XylT transmembrane domain) to map the subcellular sites of processing . This approach revealed that while SBT6.1 performs initial processing in the Golgi, additional maturation steps occur in other compartments or the extracellular space.

How evolutionarily conserved is SBT6.1 function across species?

SBT6.1 is notable for its high evolutionary conservation, being one of only two Arabidopsis SBTs that originated before the divergence of Metazoa and Viridiplantae . This conservation suggests fundamental roles in cellular processes. Researchers can investigate this evolutionary conservation using several approaches:

• Comparative sequence analysis:

  • Align SBT6.1 sequences from diverse plant species and metazoans

  • Identify conserved catalytic domains and regulatory regions

  • Map conservation onto structural models to identify functional constraints

• Cross-species complementation studies:

  • Express SBT6.1 orthologs from different species in Arabidopsis sbt6.1 mutants

  • Use SBT6.1 antibodies to confirm expression

  • Assess functional rescue of phenotypes (if antibody cross-reacts with orthologs)

• Comparative substrate specificity:

  • Test substrate processing by SBT6.1 orthologs from different species

  • Use in vitro cleavage assays with recombinant substrates

  • Compare processing patterns and efficiency

The significant expansion of the SBT family in plants involved both whole genome and tandem gene duplications with differential neo- and sub-functionalization resulting in many taxon-specific clades . SBT6.1's conservation suggests it performs a core cellular function that predates the plant-animal divergence. When planning comparative studies, researchers should assess whether available SBT6.1 antibodies cross-react with orthologs from other species, or whether species-specific antibodies are required.

What could cause weak or no signal when using SBT6.1 antibody in Western blot?

Several factors can lead to weak or absent signals when detecting SBT6.1 by Western blot:

When troubleshooting, always include a positive control sample known to express SBT6.1 (such as Arabidopsis seedlings) and verify protein transfer with reversible staining methods before antibody incubation. Given that SBT6.1 is a membrane-bound protein primarily localized to the Golgi , enrichment of membrane fractions before Western blotting can significantly improve detection sensitivity.

How can I validate antibody specificity for SBT6.1?

Validating antibody specificity is crucial for obtaining reliable results. For SBT6.1 antibodies, consider these validation approaches:

• Genetic validation:

  • Compare immunoblot signals between wild-type and sbt6.1 knockout/knockdown plants

  • The absence or reduction of signal in mutant lines provides strong evidence of specificity

• Recombinant protein controls:

  • Express recombinant SBT6.1 (full-length or fragments)

  • Use as positive controls in Western blots

  • Compare migration pattern with endogenous protein

• Peptide competition assay:

  • Pre-incubate antibody with excess immunogenic peptide

  • Compare signal with non-blocked antibody

  • Specific signals should be eliminated or reduced

• Multiple antibody validation:

  • If available, use multiple antibodies raised against different epitopes

  • Concordant detection patterns support specificity

• Mass spectrometry validation:

  • Immunoprecipitate proteins using SBT6.1 antibody

  • Identify pulled-down proteins by mass spectrometry

  • Confirm presence of SBT6.1 peptides

Remember that SBT6.1 belongs to a family with 56 members in Arabidopsis , so cross-reactivity with closely related family members is a potential concern. Check sequence similarity of the immunogen (amino acids 175-473) with other family members to assess potential cross-reactivity risks.

What is the relationship between SBT6.1 and CLEL peptide maturation?

SBT6.1 plays a critical role in the maturation pathway of CLEL/GOLVEN signaling peptides, which control cell elongation in Arabidopsis . The relationship between SBT6.1 and CLEL peptide maturation involves:

• Initial processing in the secretory pathway:

  • SBT6.1 performs the first processing step of CLEL precursors in the Golgi

  • This cleavage is necessary but not sufficient for producing mature, bioactive peptides

  • SBT6.1 processing sites are located considerably upstream of the final mature peptide

• Multi-step maturation process:

  • After SBT6.1 cleavage, additional processing at the N-terminus is required for complete maturation

  • Pre-processed precursors continue through the secretory pathway

  • Final maturation may occur in later compartments or the extracellular space

• Regulatory control:

  • SBT6.1 activity is regulated by the Serpin1 inhibitor

  • This creates a complex regulatory network controlling the availability of bioactive peptides

  • The interaction between SBT6.1 and Serpin1 has been confirmed by tandem affinity purification

Research using deletion constructs lacking SBT6.1 processing sites has demonstrated that these sites are essential for proper CLEL maturation . When studying this pathway, researchers should consider the sequential nature of processing events and the fact that SBT6.1 performs a pre-activation step rather than producing the final mature peptide directly.

How can switchable antibody complex technology be applied to SBT6.1 research?

Recent developments in switchable antibody complex technology, such as the LITE (Ligand-Induced T-cell Engager) platform described in search result , could potentially be adapted for SBT6.1 research to create novel experimental tools:

• Inducible SBT6.1 inhibition:

  • Design a system where one antibody component targets SBT6.1

  • The other component could carry an inhibitory domain

  • Small molecule inducer would bring these components together to inhibit SBT6.1 activity in a temporally controlled manner

• Spatiotemporal tracking of SBT6.1 activity:

  • Create a split reporter system where one component recognizes SBT6.1

  • The other component carries a fluorescent or enzymatic reporter

  • Small molecule inducer would enable visualization of SBT6.1 localization with temporal control

• Targeted SBT6.1 degradation:

  • Adapt the technology to deliver ubiquitin ligase components to SBT6.1

  • Enable inducible, rapid depletion of endogenous SBT6.1 protein

  • Provide an alternative to genetic knockouts for studying acute loss of function

The LITE system's key advantage is enabling "rapid, reversible, and tunable assembly of functional antibody complexes using a small-molecule dimerizer" . This would allow researchers to manipulate SBT6.1 function with precise temporal control, overcoming limitations of conventional genetic approaches where compensatory mechanisms may obscure phenotypes.

While this represents an advanced application not yet reported for SBT6.1 research, the principles demonstrated in the development of switchable bispecific T-cell engagers (bsTCEs) could be adapted for studying plant proteases in novel ways .