Cysteine protease inhibitor 4 Antibody

What is Cysteine protease inhibitor 4 Antibody and what specific target does it recognize?

Cysteine protease inhibitor 4 Antibody is a polyclonal antibody raised in rabbits against recombinant Solanum tuberosum (Potato) Cysteine protease inhibitor 4 protein. It specifically targets the cysteine protease inhibitor 4 protein (UniProt No. P58602) . This antibody has been produced through immunization protocols followed by antigen affinity purification to ensure specificity and sensitivity in detecting its target protein .

The antibody is provided in liquid form with a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . It is designed specifically for research applications and should not be used for diagnostic or therapeutic purposes .

What are the optimal storage and handling conditions for maintaining antibody activity?

For optimal preservation of antibody activity, the following storage conditions should be observed:

• Upon receipt, store the antibody at -20°C or -80°C

• Avoid repeated freeze-thaw cycles which can significantly reduce antibody functionality

• When working with the antibody, keep it on ice and return to appropriate storage conditions promptly

• For long-term storage, consider aliquoting the antibody into smaller volumes to minimize freeze-thaw cycles

What validated applications can Cysteine protease inhibitor 4 Antibody be used for?

The Cysteine protease inhibitor 4 Antibody has been validated for the following research applications:

These applications enable researchers to study the expression, abundance, and molecular characteristics of cysteine protease inhibitor 4 protein in plant samples . The antibody specifically ensures identification of the antigen in these applications , making it a reliable tool for studying this important plant defense protein.

How does species reactivity affect experimental design when using this antibody?

The Cysteine protease inhibitor 4 Antibody has been specifically developed to react with cysteine protease inhibitor 4 from Solanum tuberosum (Potato) . This species specificity has important implications for experimental design:

• The antibody is optimized for detecting potato cysteine protease inhibitor 4

• Cross-reactivity with homologous proteins from other plant species has not been fully characterized

• When studying other Solanum species or more distant plant relatives, preliminary validation experiments should be conducted

• Control experiments using potato samples should be included when testing reactivity in other species

• Researchers studying non-potato species should consider alternative antibodies specific to their species of interest

Understanding these species reactivity limitations is crucial when designing experiments and interpreting results, particularly in comparative studies across different plant species.

What controls should be included when validating this antibody for specific experimental systems?

Proper validation of the Cysteine protease inhibitor 4 Antibody requires inclusion of appropriate controls:

These controls help establish the specificity, sensitivity, and reliability of the antibody in your specific experimental system. Proper validation is essential for generating reproducible and trustworthy research data, particularly when publishing findings in peer-reviewed journals.

How can researchers effectively use Cysteine protease inhibitor 4 Antibody to study plant defense mechanisms?

Cysteine protease inhibitors play critical roles in plant defense against pathogens and herbivores. To effectively leverage this antibody in defense studies:

• Expression profiling during pathogen challenge: Monitor temporal changes in cysteine protease inhibitor 4 expression following pathogen infection using Western blot or ELISA techniques .

• Comparative analysis across cultivars: Compare constitutive and induced expression of the inhibitor between resistant and susceptible cultivars to identify correlations with disease resistance.

• Co-localization studies: Combine this antibody with pathogen-specific markers to investigate spatial relationships between the inhibitor and invading organisms during infection.

• Defense signaling pathway analysis: Use this antibody to monitor inhibitor expression following treatment with defense signaling molecules (e.g., jasmonic acid, salicylic acid) to determine regulatory pathways.

• Protein-protein interaction studies: Employ co-immunoprecipitation approaches to identify which proteases are targeted by the inhibitor during defense responses.

This research approach parallels strategies used to study cysteine protease inhibitors in other systems, where targeting proteases effectively disrupts pathogen survival and infection processes .

What methodological approaches can optimize Western blot detection using this antibody?

Optimizing Western blot protocols for Cysteine protease inhibitor 4 Antibody requires systematic adjustment of several parameters:

• Sample preparation optimization:

  • Extract proteins using buffers containing multiple protease inhibitors to prevent degradation

  • Include reducing agents (DTT or β-mercaptoethanol) in sample buffer to fully denature the protein

  • Heat samples at 95°C for 5 minutes to ensure complete denaturation

• Antibody dilution optimization:

  • Test serial dilutions (typically starting at 1:500 to 1:5000) to determine optimal concentration

  • Incubate primary antibody at 4°C overnight rather than at room temperature for better specificity

  • Prepare antibody dilutions in freshly made buffer containing 1-5% blocking agent

• Signal enhancement strategies:

  • Use PVDF membranes instead of nitrocellulose for higher protein binding capacity

  • Consider signal amplification systems for low-abundance targets

  • Optimize exposure times to achieve optimal signal-to-noise ratio

• Background reduction techniques:

  • Increase washing duration and frequency (5-6 washes of 10 minutes each)

  • Add 0.1-0.3% Tween-20 to wash buffers to reduce non-specific binding

  • Consider using specialized blocking reagents designed to reduce background

These methodological refinements should be systematically tested and documented to establish optimal conditions for specific detection of cysteine protease inhibitor 4 protein in your experimental system.

How do the mechanisms of plant cysteine protease inhibitors compare with other types of protease inhibitors?

Understanding the mechanistic differences between protease inhibitor types is crucial for experimental design:

Research has demonstrated that propeptide-based inhibitors, such as those derived from procathepsin B, can be genetically fused into antibody structures to create highly specific inhibitors with improved pharmacokinetic properties . This innovative approach demonstrates the potential for combining different inhibitory mechanisms to create more effective research and therapeutic tools.

What techniques can be used to study the interaction between cysteine protease inhibitors and their target proteases?

Multiple complementary techniques can characterize inhibitor-protease interactions:

• Enzyme kinetics analysis:

  • Measure inhibition constants (Ki) using purified components

  • Determine inhibition mechanism (competitive, non-competitive, or uncompetitive)

  • Compare second-order rate constants across different proteases to quantify specificity

• Structural biology approaches:

  • X-ray crystallography of inhibitor-protease complexes

  • NMR studies to map binding interfaces

  • Molecular docking simulations to predict binding modes

• Cellular trafficking studies:

  • Track protease and inhibitor localization using immunofluorescence

  • Monitor accumulation patterns in subcellular compartments

  • Investigate trafficking pathways similar to those observed with lysosomal proteases

• Activity-based protein profiling:

  • Use activity-based probes to label active proteases

  • Determine how inhibitor presence affects protease labeling patterns

  • Combine with mass spectrometry for protease identification

• Bioengineering approaches:

  • Create fusion constructs between inhibitors and reporter proteins

  • Monitor protein-protein interactions in real-time

  • Design structure-guided mutations to alter inhibitor specificity

These techniques have revealed important insights about inhibitor mechanisms, including how synthetic inhibitors can selectively target pathogen proteases while sparing host enzymes , a finding that has implications for both research tools and therapeutic development.

How can researchers troubleshoot non-specific binding and background issues when using this antibody?

Non-specific binding is a common challenge that can be addressed through systematic optimization:

• Blocking optimization:

  • Test different blocking agents (BSA, casein, non-fat dry milk)

  • Increase blocking time (from 1 hour to overnight)

  • Consider commercial blocking solutions designed to reduce background

• Antibody dilution refinement:

  • Prepare more dilute antibody solutions (1:1000 to 1:10,000 range)

  • Use freshly prepared antibody dilutions for each experiment

  • Dilute antibody in buffer containing 1-5% of the blocking agent

• Pre-adsorption techniques:

  • Incubate diluted antibody with proteins from non-target species

  • Remove complexes by centrifugation before using the antibody

  • This can effectively reduce cross-reactivity with conserved epitopes

• Buffer modification strategies:

  • Increase salt concentration in wash buffers (up to 500mM NaCl)

  • Add low concentrations of non-ionic detergents (0.1-0.3% Tween-20)

  • Consider adding low concentrations of competing proteins

• Signal-to-noise optimization:

  • Reduce primary antibody incubation time

  • Increase washing duration and frequency

  • Optimize detection reagent concentration and exposure times

Systematic documentation of these optimization steps will help establish reliable protocols that maximize specific signal while minimizing background interference.

What are the current research applications linking cysteine protease inhibitors to disease resistance mechanisms?

Cysteine protease inhibitors are increasingly recognized as key components in disease resistance mechanisms:

• Pathogen virulence suppression:

  • Cysteine proteases are key virulence factors in many pathogens

  • Plant inhibitors can directly target these proteases to suppress infection

  • Similar mechanisms have been demonstrated in studies of parasitic infections like Leishmaniasis

• Programmed cell death regulation:

  • Cysteine proteases often function in programmed cell death pathways

  • Inhibitors can modulate hypersensitive response during infection

  • This regulation affects disease progression and containment

• Structural defense fortification:

  • Inhibitors can protect cell wall integrity from pathogen-secreted proteases

  • This physical barrier maintenance limits pathogen spread

  • Similar protective effects have been observed in other host-pathogen systems

• Signaling pathway modulation:

  • Protease inhibitors can regulate defense signaling cascades

  • This modulation affects timing and magnitude of defense responses

  • Understanding these pathways provides targets for enhancing resistance

• Transgenic resistance engineering:

  • Overexpression of cysteine protease inhibitors can enhance resistance

  • This approach parallels genetic deletion studies that demonstrated reduced pathogen survival

  • Multiple inhibitor combinations may provide more durable resistance

Research on microbial and parasitic systems has demonstrated that cysteine protease inhibitors can effectively kill pathogens at concentrations that do not affect host cells , suggesting potential applications in both research and disease management strategies.

How can Cysteine protease inhibitor 4 Antibody be integrated with other techniques for comprehensive protease activity profiling?

A multi-technique approach provides the most comprehensive analysis of protease regulation:

• Functional proteomics integration:

  • Combine antibody detection with activity-based protein profiling

  • Correlate protease inhibitor expression with active protease populations

  • Identify post-translational modifications affecting inhibitory activity

• Systems biology approaches:

  • Create comprehensive models of protease-inhibitor networks

  • Map expression changes across developmental stages or stress conditions

  • Integrate transcriptomic and proteomic data to identify regulatory nodes

• Subcellular localization studies:

  • Perform co-localization analysis of inhibitors and target proteases

  • Track trafficking patterns in response to stress conditions

  • Similar approaches have revealed key insights about protease localization in disease models

• Real-time monitoring systems:

  • Develop fluorescent reporter systems for protease activity

  • Monitor inhibitor effects on protease activity in living cells

  • Create dynamic models of inhibitor function during stress responses

• Structural biology integration:

  • Determine crystal structures of inhibitor-protease complexes

  • Design improved inhibitors based on structural insights

  • This approach has been successful in developing humanized antibody inhibitors

• Translation to field applications:

  • Develop high-throughput screening methods for resistance breeding

  • Create diagnostic tools to monitor protease activity in field conditions

  • Bridge laboratory findings with practical agricultural applications

The combination of these techniques allows researchers to build comprehensive models of how cysteine protease inhibitors function in complex biological systems, similar to the multi-faceted approaches that have successfully elucidated the role of cysteine protease inhibitors in other systems .