HSP90-6 Antibody

What is HSP90-6 and how does it differ from other HSP90 isoforms?

HSP90-6 is a specific isoform of the heat shock protein 90 family involved in protein folding and cellular stress responses. While HSP90 proteins generally function as molecular chaperones regulating signaling pathways and correcting misfolded proteins, HSP90-6 appears to have specific roles in nutrient metabolism, particularly in plant systems. Research has shown that HSP90-6 is involved in grain filling via carbon and nitrogen metabolism in maize . It differs from other commonly studied isoforms like HSP90α (HSP90AA1) and HSP90β (HSP90AB1) which share approximately 90% sequence identity with each other but have distinct functions and expression patterns .

What are the primary applications for HSP90-6 antibodies in research?

HSP90-6 antibodies are valuable tools for multiple research applications including:

• Western blotting for protein expression analysis

• Immunohistochemistry (IHC) for tissue localization

• Immunofluorescence (IF) for subcellular localization

• Immunoprecipitation (IP) for protein-protein interaction studies

• Co-immunoprecipitation (Co-IP) for complex formation analysis

• RNA immunoprecipitation (RIP) for RNA-protein interactions

These applications allow researchers to investigate HSP90-6 expression patterns, cellular distribution, and functional interactions with client proteins in various experimental systems.

How should I validate the specificity of an HSP90-6 antibody?

To validate HSP90-6 antibody specificity:

• Perform Western blotting with positive and negative control samples

• Include recombinant HSP90-6 protein as a reference standard

• Test cross-reactivity against other HSP90 isoforms (particularly HSP90α and HSP90β)

• Use knockout or knockdown samples as negative controls

• Verify results using multiple antibodies targeting different epitopes of HSP90-6

• Conduct peptide competition assays to confirm binding specificity

In particular, HSP90 antibodies should be carefully validated to distinguish between the highly homologous isoforms, as seen in validation studies where researchers include recombinant HSP90α and HSP90β proteins for reference .

What are the optimal dilutions for different applications of HSP90-6 antibodies?

Note: These dilutions should be optimized for each specific antibody and experimental system. The appropriate dilution is sample-dependent and should be determined empirically for optimal results .

What is the recommended protocol for Western blotting with HSP90-6 antibodies?

For optimal Western blotting results with HSP90-6 antibodies:

• Prepare protein lysates in an appropriate buffer (RIPA or NP-40 based buffers)

• Load 10-30 μg of total protein per lane

• Separate proteins using 8-10% SDS-PAGE (HSP90 proteins appear at ~85-90 kDa)

• Transfer to PVDF membrane

• Block with 5% non-fat milk or BSA in TBST

• Incubate with primary HSP90-6 antibody at optimized dilution (typically 1:5000-1:50000) overnight at 4°C

• Wash with TBST (3-5 times, 5-10 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence

• For loading control, strip and reprobe with antibodies against housekeeping proteins like α-tubulin or GAPDH

This protocol has been validated in multiple cell lines including human (HeLa, LNCaP, HEK-293, MCF-7, Jurkat), rat (HSC-T6, ROS1728), and mouse (NIH/3T3, 4T1) models .

What antigen retrieval methods are recommended for immunohistochemistry with HSP90-6 antibodies?

For optimal immunohistochemical detection of HSP90-6:

• Section formalin-fixed, paraffin-embedded tissues at 4-6 μm thickness

• Primary antigen retrieval recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

• Heat-induced epitope retrieval is typically more effective than enzymatic retrieval

• Apply primary antibody at 1:50-1:500 dilution

• Incubate overnight at 4°C or 1-2 hours at room temperature

• Use appropriate detection system (e.g., HRP-polymer or avidin-biotin complex)

• Counterstain, dehydrate, and mount

The chosen antigen retrieval method should be optimized based on the specific tissue type and fixation conditions.

How can I use HSP90-6 antibodies to study its role in nutrient metabolism?

To investigate HSP90-6's role in nutrient metabolism, particularly in plant systems:

• Perform comparative expression analysis between wild-type and HSP90.6 mutant tissues

• Use Western blotting to quantify HSP90-6 protein levels at different developmental stages

• Conduct immunohistochemistry to localize HSP90-6 in specific tissues like endosperm and embryo

• Apply co-immunoprecipitation to identify HSP90-6 interactions with carbon and nitrogen metabolism enzymes

• Combine with transcriptomic analysis to identify downstream gene expression changes in amino acid biosynthesis and carbon metabolism pathways

• Use in situ hybridization in parallel with immunofluorescence to correlate transcript and protein localization

Research has shown that HSP90.6 is highly expressed in early stages of grain development and its expression pattern changes throughout development, being highly distributed in kernels at 5 days after pollination (DAP) and present in embryo and endosperm at 12 DAP .

What considerations are important when using HSP90-6 antibodies in cancer research?

When applying HSP90-6 antibodies in cancer research:

• Verify antibody specificity for HSP90 isoforms relevant to your cancer model

• Consider differential expression of HSP90 isoforms in various cancer types

• Evaluate cell surface versus intracellular HSP90 expression (extracellular HSP90 is significantly higher in tumor cells than normal cells)

• Correlate HSP90 expression with client oncoproteins and tumor progression markers

• When studying HSP90 inhibitors, examine effects on multiple signaling pathways simultaneously

• Consider using HSP90 antibodies to monitor therapy response in cancer models

HSP90 is critically involved in cancer biology by interacting with oncogenic client proteins, making it a potential therapeutic target. Plasma concentrations of HSP90 have been shown to positively correlate with tumor malignancy in cancer patients .

How can I differentiate between HSP90 isoforms using antibodies?

To differentiate between highly homologous HSP90 isoforms:

• Select antibodies raised against unique epitopes, particularly targeting the N-terminal regions where sequence divergence is greatest

• Verify specificity using recombinant proteins of each isoform

• Perform Western blotting with controls expressing individual isoforms

• Consider using antibodies raised against synthetic peptides corresponding to isoform-specific regions

• Note that HSP90α (HSP90AA1) and HSP90β (HSP90AB1) share 90% identity but have distinguishable sequences, particularly at the N-terminus

• Include appropriate positive controls (e.g., PA3-013 specifically detects HSP90α/HSP86 but not HSP84)

For example, the PA3-013 antibody was raised against a synthetic peptide corresponding to residues P(2) E E T Q T Q D Q P M(12) of mouse HSP86, a region where the N-terminal sequences of HSP84 and HSP86 show the largest differences .

Why might I observe multiple bands when using HSP90-6 antibodies in Western blotting?

Multiple bands in Western blotting with HSP90-6 antibodies may occur due to:

• Post-translational modifications like phosphorylation or acetylation

• Cross-reactivity with other HSP90 isoforms (HSP90α, HSP90β)

• Proteolytic degradation during sample preparation

• Alternative splice variants of HSP90

• Non-specific binding to related heat shock proteins

To address this issue:

• Include recombinant HSP90 isoforms as reference standards

• Optimize lysis buffer composition with appropriate protease inhibitors

• Test different blocking agents (BSA vs. non-fat milk)

• Verify antibody specificity using knockout or knockdown controls

• Consider preabsorption with potential cross-reactive proteins

HSP90 proteins typically appear at approximately 85-90 kDa molecular weight range on Western blots, with some variation depending on post-translational modifications and isoform .

How can I optimize immunoprecipitation experiments with HSP90-6 antibodies?

For successful immunoprecipitation of HSP90-6:

• Use mild lysis buffers (e.g., NP-40 based) to preserve protein-protein interactions

• Add 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Pre-clear lysates with appropriate control IgG and protein A/G beads

• Include both positive (known HSP90-6 expressing) and negative control samples

• Optimize incubation time and temperature (typically overnight at 4°C)

• For co-immunoprecipitation, consider crosslinking to stabilize transient interactions

• Use appropriate elution conditions to preserve co-precipitated proteins

• Control for non-specific binding using isotype-matched control antibodies

Immunoprecipitation experiments have demonstrated that HSP90 exists primarily as homodimers in cells and can be precipitated when complexed with other proteins such as the aryl hydrocarbon receptor .

What control samples should I include when using HSP90-6 antibodies?

Essential controls for HSP90-6 antibody experiments include:

• Positive control: Cells/tissues known to express HSP90-6 (e.g., HeLa, HEK-293, Jurkat cells for mammalian HSP90)

• Negative control:

  • Isotype-matched irrelevant antibody

  • HSP90-6 knockout or knockdown samples

  • Tissues from relevant mutant models (e.g., hsp90.6 mutant in plant studies)

• Loading control: Housekeeping proteins like α-tubulin, GAPDH, or β-actin

• Specificity controls:

  • Peptide competition assay

  • Recombinant HSP90 isoforms

• Technical controls:

  • Secondary antibody only

  • Blocking reagent optimization

For instance, research on maize HSP90.6 utilized hsp90.6 knockout mutant and single-amino acid mutant (ehsp90.6) as controls to validate antibody specificity and functional studies .

What is known about HSP90-6's role in plant development?

Research on HSP90.6 in plants has revealed:

• HSP90.6 regulates nutrient metabolism in maize grain development

• A single-amino acid mutation in the HATPase_c domain reduces ATPase activity, resulting in smaller grains

• Functional loss of HSP90.6 downregulates expression of amino acid biosynthesis and carbon metabolism-related genes

• HSP90.6 mutants show differences in kernel development as early as 10 days after pollination (DAP)

• HSP90.6 is highly expressed in early grain development stages

• Expression patterns change during development:

  • Highly and evenly distributed in kernels at 5 DAP

  • Strong in embryo and endosperm at 12 DAP

  • Low levels by 20 DAP except in scutellum

These findings suggest HSP90.6 plays a critical role in grain filling and development in maize, with potential implications for crop improvement.

How are HSP90 inhibitors being developed as potential therapeutic agents?

HSP90 inhibitors are being developed as therapeutic agents through several approaches:

• Targeting mechanisms:

  • N-terminal domain (NTD) inhibitors targeting ATP-binding site

  • C-terminal domain (CTD) inhibitors

  • Isoform-selective inhibitors

• Current challenges:

  • Drug resistance development

  • Dose-limiting toxicity

  • Poor pharmacokinetic profiles

• Research strategies:

  • Combination therapies with chemotherapy agents

  • Co-administration with targeted therapies

  • Integration with immunotherapy approaches

• Progress:

  • Numerous inhibitors have been reported with potential as cancer-targeted therapies

  • Preclinical and clinical trials are ongoing

  • None have yet been approved for clinical treatment

Research indicates that HSP90 inhibition suppresses oncogenic pathways in cancer cells by interrupting the ATPase activity of HSP90, making it a promising target for targeted cancer therapies .

What recent advances have been made in understanding HSP90's extracellular functions?

Recent advances in understanding extracellular HSP90 functions include:

• Differential expression: Cell surface HSP90 expression is considerably higher in tumor cells compared to normal cells

• ATP independence: Extracellular HSP90 can function independently of ATP to adapt to reduced ATP-extracellular environment

• Secretion patterns: Tumor cells secrete HSP90 consecutively, while healthy cells secrete it only under stress conditions

• Clinical correlation: Plasma concentrations of HSP90 positively associate with tumor malignancy in cancer patients

• Therapeutic potential: Blocking or neutralizing HSP90 secretion can inhibit cancer invasion and migration

• Functional discoveries: HSP90 drives functional heme maturation of inducible nitric oxide synthase (iNOS) and soluble guanylate cyclase (sGC)

These findings highlight extracellular HSP90 as a potential therapeutic target, particularly for preventing malignant tumor progression through inhibition of its secretion or neutralization of its extracellular functions .