HSP90-5 Antibody

What is HSP90-5 and what does HSP90-5 antibody detect?

HSP90-5 refers to the chloroplast-localized HSP90 family molecular chaperone (also designated as HSP90.5 in Arabidopsis), which plays essential roles in plant abiotic stress resistance, photomorphogenesis, and nuclear-encoded protein import into the chloroplast . The HSP90-5 antibody specifically recognizes this chloroplast isoform of HSP90, which is distinct from cytosolic HSP90 variants. In Arabidopsis studies, anti-HSP90.5 antibody has been shown to recognize both endogenous HSP90.5 and FLAG-tagged HSP90.5 versions .

HSP90 proteins are approximately 90 kDa molecular weight chaperones that are induced under stress conditions but are also among the most abundant cellular proteins under normal conditions . They associate with numerous intracellular proteins, including steroid receptors, actin, tubulin, and various kinases, maintaining their structural integrity and facilitating proper function .

What are the primary applications of HSP90-5 antibody in research?

HSP90-5 antibody can be utilized across multiple experimental platforms:

• Western Blotting (WB): For detecting and quantifying HSP90-5 protein expression levels in tissue or cell lysates. This application allows researchers to monitor changes in HSP90-5 expression under different experimental conditions .

• Immunohistochemistry (IHC): For visualizing the localization and distribution of HSP90-5 in fixed tissue sections, particularly useful for studying its chloroplast localization in plant tissues .

• Immunofluorescence (IF): For examining subcellular localization and co-localization with other proteins in both fixed and permeabilized cells .

• Flow Cytometry (FC): For analyzing HSP90-5 expression in individual cells within heterogeneous populations .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of HSP90-5, including assessing extracellular HSP90 secretion by cells .

• Immunoprecipitation: For isolating HSP90-5 and its associated protein complexes from cell or tissue lysates .

How can I validate the specificity of HSP90-5 antibody in my experimental system?

Validating antibody specificity is crucial for ensuring reliable research results. For HSP90-5 antibody, consider these validation approaches:

• Positive and Negative Controls: Use tissues or cell lines known to express or not express HSP90-5. For plant studies, compare wild-type plants with HSP90.5 cosuppression or knockout lines .

• Western Blot Analysis: Confirm detection of a single band of the expected molecular weight (~90 kDa). The Arabidopsis HSP90.5 appears as a 90 kDa protein on immunoblots .

• Antibody Specificity Testing: Verify that the HSP90-5 antibody does not cross-react with cytosolic HSP90 isoforms. In Arabidopsis studies, anti-HSP90.5 antibody specifically recognized chloroplast HSP90.5 without cross-reacting with cytosolic HSP90.1-4 isoforms .

• Recombinant Protein: Use purified recombinant HSP90-5 as a positive control. Purified recombinant fragment of human HSP90AA1 expressed in E. coli has been used as an immunogen for HSP90 antibody production .

• Genetic Validation: When possible, use genetic approaches like RNAi knockdown or CRISPR-Cas9 knockout of HSP90-5 to confirm antibody specificity through loss of signal .

What are the optimal storage and handling conditions for HSP90-5 antibody?

To maintain optimal antibody performance:

• Storage Temperature: Store antibodies at -80°C for long-term storage to preserve activity .

• Buffer Composition: HSP90 antibodies are typically stored in PBS-only buffer or with stabilizing proteins such as BSA .

• Aliquoting: Upon receipt, aliquot antibodies to avoid repeated freeze-thaw cycles, which can denature proteins and reduce antibody efficiency.

• Working Dilutions: Determine optimal working dilutions for each application through titration experiments. Typical working dilutions range from 1:500 to 1:2000 for Western blotting and 1:100 to 1:500 for immunostaining applications.

• Reconstitution: For lyophilized antibodies, reconstitute in sterile water or the recommended buffer, ensuring complete dissolution before use.

How can I distinguish between different HSP90 isoforms using antibodies?

Distinguishing between HSP90 isoforms requires careful antibody selection and experimental design:

• Isoform-Specific Epitopes: Select antibodies raised against unique regions of specific HSP90 isoforms. For example, antibodies recognizing the N-terminal region of HSP90.5 can distinguish it from cytosolic HSP90 isoforms .

• Expression System Validation: When studying tagged HSP90 variants, verify that the antibody can recognize both endogenous and tagged forms. In Arabidopsis studies, anti-HSP90.5 antibody recognized both endogenous and FLAG-tagged HSP90.5, although they were difficult to distinguish due to similar molecular weights (the FLAG-tagged version being only 8 amino acids longer) .

• Comparative Western Blot Analysis: Run parallel blots with isoform-specific antibodies. For example, use anti-HSP90.5 antibody alongside anti-HSP90.2 antibody to differentiate chloroplast and cytosolic HSP90 expression patterns .

• Genetic Controls: Utilize tissues from knockout or knockdown models for specific HSP90 isoforms as negative controls.

• Subcellular Fractionation: Isolate different cellular compartments (cytosol, chloroplast, etc.) before immunoblotting to enrich for specific HSP90 isoforms based on their known localization patterns .

What methodological approaches can I use to study extracellular HSP90 using antibodies?

Extracellular HSP90 (eHSP90) plays important roles in cancer progression and other pathological processes. To study eHSP90:

• Culture Media Concentration: Concentrate cell culture media using ultrafiltration devices to enrich for secreted proteins before detection .

• Sandwich ELISA: Develop a sandwich ELISA using HSP90-5 antibody as a capture antibody immobilized on plates. This approach has been successfully used to detect eHSP90 secreted by breast cancer cell lines like MDA-MB-231 and MDA-MB-453 .

• Surface Plasmon Resonance (SPR): Use SPR to detect and quantify eHSP90 in culture media. Immobilize anti-HSP90 antibody fragments (such as scFv) on SPR chips to capture eHSP90 from biological samples .

• Comparative Analysis: Include non-cancer cell lines (like human skin fibroblast BJ cells) as negative or low-expression controls when analyzing eHSP90 secretion, as they typically show significantly lower eHSP90 secretion compared to cancer cell lines .

• Western Blot Verification: Confirm eHSP90 secretion by Western blotting of concentrated culture media, noting that secreted HSP90 is often a C-terminal truncated form compared to intracellular HSP90 .

How can I optimize immunoprecipitation protocols using HSP90-5 antibody to study chaperone-client interactions?

Studying HSP90-5 interactions with client proteins requires optimized immunoprecipitation (IP) protocols:

• Cross-linking Optimization: Determine optimal cross-linking conditions to stabilize transient HSP90-client interactions. Test various cross-linkers (e.g., DSP, formaldehyde) with different incubation times.

• Lysis Buffer Selection: Use gentle lysis buffers (containing 0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions. Include ATP (1-5 mM) in buffers to stabilize HSP90-client complexes, as ATP binding affects HSP90 conformation and client binding .

• Co-chaperone Considerations: Add appropriate co-chaperones or inhibit their function to study their impact on HSP90-client interactions. HSP90 functions with numerous co-chaperones that modulate its activity and client specificity.

• Sequential IP: Perform sequential IPs (first with anti-HSP90-5, then with antibodies against suspected client proteins) to verify specific interactions and reduce background.

• Mass Spectrometry Analysis: Combine IP with mass spectrometry to identify novel HSP90-5 client proteins and interaction partners. This approach can reveal the HSP90-5 interactome in different biological contexts.

What are the key considerations when using HSP90-5 antibody to study chloroplast development and biogenesis?

When investigating chloroplast development using HSP90-5 antibody:

• Developmental Stage Sampling: HSP90.5 expression can be developmentally regulated, with increasing expression from earlier to later initiated leaves in Arabidopsis. Carefully select and document developmental stages of tissues used for analysis .

• Tissue-Specific Expression: HSP90.5 cosuppression in transgenic plants can occur in a tissue-specific manner, primarily affecting late developmental stages in adult leaves and inflorescence tissues. Use tissues from different developmental stages to comprehensively assess HSP90.5 function .

• Chloroplast Isolation: Optimize chloroplast isolation protocols to study HSP90.5 in its native organellar context. Verify proper chloroplast localization of HSP90.5 using fractionation quality controls .

• Protein Complex Analysis: Use size exclusion chromatography to analyze HSP90.5 dimerization and complex formation. HSP90 functions as a dimer, and dimerization state can affect its chaperone activity .

• Electron Microscopy Correlation: Combine HSP90.5 expression analysis with transmission electron microscopy to correlate HSP90.5 levels with chloroplast structural integrity and development. Studies have shown that chloroplast development is significantly impaired in HSP90.5 cosuppression lines .

How can HSP90-5 antibody be used in developing targeted anticancer strategies?

HSP90 has emerged as an important target for anticancer therapies, and antibodies can play a role in research and therapeutic development:

• Extracellular HSP90 Targeting: Generate and characterize antibody fragments (scFvs) that specifically target extracellular HSP90. These could potentially block eHSP90's role in cancer metastasis without affecting intracellular HSP90 functions .

• Affinity Maturation: Implement affinity maturation procedures to enhance the binding properties of HSP90-specific antibody fragments. Techniques like "off-rate selection" can improve scFv affinity to target proteins .

• Diagnostic Applications: Develop ELISA or other immunoassays using HSP90-5 antibody to quantify plasma HSP90 levels, which have been correlated with tumor malignancy in clinical cancer patients .

• Experimental Design Considerations: When investigating HSP90 in cancer contexts, include appropriate cellular models. For example, breast cancer cell lines such as MDA-MB-231 and MDA-MB-453 have been used to study eHSP90 secretion, while human skin fibroblast cell lines serve as non-cancer controls .

• Therapeutic Antibody Development: Study the potential of HSP90-specific antibodies to deliver drugs or imaging agents specifically to cancer cells with elevated HSP90 expression or secretion.

What are common issues with HSP90-5 antibody in Western blotting and how can they be resolved?

When performing Western blotting with HSP90-5 antibody:

• Multiple Bands: If detecting multiple bands, optimize primary antibody concentration or blocking conditions. With properly optimized conditions, anti-HSP90.5 antibody should detect a single band at approximately 90 kDa .

• Poor Signal: If experiencing weak signal:

  • Increase protein loading (20-50 μg total protein is typically sufficient)

  • Optimize antibody concentration through titration experiments

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

• High Background: If background is excessive:

  • Increase blocking time or blocker concentration

  • Add 0.1-0.3% Tween-20 to wash buffers

  • Dilute primary antibody further

  • Use more stringent washing (increase number and duration of washes)

• Distinguishing Tagged vs. Untagged Proteins: When working with tagged HSP90.5 (e.g., FLAG-tagged), carefully control sample loading and exposure times during immunoblotting to visualize the slight size difference between endogenous and tagged proteins .

How can I optimize immunofluorescence protocols using HSP90-5 antibody?

For optimal immunofluorescence results:

• Fixation Method: Test different fixation methods (4% paraformaldehyde, methanol, or acetone) to determine which best preserves HSP90-5 epitopes while maintaining cellular structure.

• Permeabilization: Optimize permeabilization conditions (0.1-0.5% Triton X-100 or 0.05-0.1% saponin) to ensure antibody access to intracellular HSP90-5 while minimizing background.

• Antigen Retrieval: If working with fixed tissues or cells with masked epitopes, evaluate antigen retrieval methods (heat-induced or enzymatic) to enhance signal.

• Blocking Strategy: Use 5-10% normal serum from the same species as the secondary antibody to reduce non-specific binding.

• Antibody Concentration: Titrate primary antibody concentration (typically 1:50 to 1:500) to determine optimal signal-to-noise ratio.

• Controls:

  • Include a negative control omitting primary antibody

  • Use cells known to express high or low levels of HSP90-5

  • Consider including a peptide competition control to verify specificity

What methodological approaches can help distinguish endogenous from recombinant HSP90-5 in research models?

When working with both endogenous and recombinant HSP90-5:

• Tag-Specific Antibodies: Use antibodies against epitope tags (e.g., FLAG, His) to specifically detect recombinant HSP90-5. This approach was successfully used in studies with FLAG-tagged HSP90.5 in Arabidopsis .

• Size Discrimination: Carefully optimize gel separation conditions to resolve the small size differences between tagged and endogenous proteins. FLAG-tagged HSP90.5 is only 8 amino acids longer than endogenous HSP90.5 and requires careful sample loading and controlled exposure during immunoblotting to visualize as separate bands .

• Dual Antibody Approach: Use both anti-HSP90-5 and anti-tag antibodies on parallel blots or in dual-color immunofluorescence to compare localization patterns.

• Expression System Controls: Include appropriate controls:

  • Wild-type cells/tissues (expressing only endogenous HSP90-5)

  • Empty vector-transfected cells (also expressing only endogenous HSP90-5)

  • HSP90-5 knockout/knockdown models expressing only the recombinant protein

• Transgene Silencing Awareness: Be aware that transgene expression can trigger cosuppression of both the transgene and endogenous gene, as observed in Arabidopsis HSP90.5-FLAG transgenic plants. Monitor total HSP90-5 levels to detect potential cosuppression effects .