VHL PAT82B10AT Antibody

What is the VHL PAT82B10AT antibody and what is its target?

The VHL PAT82B10AT antibody is a mouse monoclonal antibody that specifically recognizes human Von Hippel-Lindau (VHL) tumor suppressor protein. This antibody is derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with a recombinant human VHL protein fragment (amino acids 1-154) purified from E. coli. It belongs to the IgG2b isotype with kappa light chains . VHL is a critical tumor suppressor protein that functions as the substrate recognition component of an E3 ubiquitin ligase complex involved in targeting proteins containing hydroxyproline residues for proteasomal degradation, most notably hypoxia-inducible factor (HIF) .

What applications has the VHL PAT82B10AT antibody been validated for?

The VHL PAT82B10AT antibody has been validated for the following applications:

• Western blot (WB) - for detecting VHL protein in denatured samples

• Enzyme-linked immunosorbent assay (ELISA) - for quantitative detection of VHL

While immunofluorescence (IF) isn't explicitly listed for the PAT82B10AT clone, other VHL antibodies have demonstrated successful application in immunohistochemistry (IHC) and immunofluorescence, suggesting potential utility in these applications following appropriate optimization .

How should the VHL PAT82B10AT antibody be stored and handled?

For optimal performance and longevity of the VHL PAT82B10AT antibody:

• Long-term storage: Store at -20°C

• Avoid repeated freeze-thaw cycles by aliquoting before freezing

• Short-term storage (1 month): 2-8°C under sterile conditions after reconstitution

• Working concentration: 1 mg/ml in PBS (pH 7.4) with 0.1% sodium azide

For long-term storage exceeding 6 months, maintaining the antibody at -20°C to -70°C is recommended to preserve activity and prevent degradation .

What controls should be included when using the VHL PAT82B10AT antibody?

For rigorous experimental design with the VHL PAT82B10AT antibody, include:

Positive controls:

• Cell lines with known VHL expression (e.g., Daudi human Burkitt's lymphoma cell line)

• Recombinant VHL protein (preferably the immunogen fragment, amino acids 1-154)

Negative controls:

• Isotype control: Mouse IgG2b isotype control antibodies at matching concentration

• Secondary antibody-only control: Omitting primary antibody to assess non-specific binding

• VHL-null or VHL-knockdown samples (when available)

Loading/technical controls:

• For Western blots: Housekeeping protein detection (β-actin, GAPDH, etc.)

• For immunofluorescence: DAPI nuclear counterstain as demonstrated in validation studies

What is the optimal concentration for using VHL PAT82B10AT antibody in different applications?

Important note: Since application requirements vary between laboratories and experimental systems, each investigation should titrate the reagent to determine optimal conditions for specific experimental settings . Start with the recommended dilution and adjust as needed based on signal-to-noise ratio.

How can the VHL PAT82B10AT antibody be used to study VHL-HIF pathway interactions?

The VHL PAT82B10AT antibody provides a valuable tool for investigating the VHL-HIF pathway through several methodological approaches:

• Co-immunoprecipitation studies:

  • Use the antibody to pull down VHL protein complexes

  • Analyze VHL interaction with HIF1α and other pathway components

  • Examine how these interactions change under different oxygen conditions

• Proteasomal degradation assays:

  • Monitor HIF1α levels in parallel with VHL detection

  • Compare normoxic vs. hypoxic conditions to assess VHL-mediated degradation

  • Use proteasome inhibitors to confirm the degradation mechanism

• E3 ligase complex analysis:

  • Detect VHL in association with other E3 ligase components (Elongin B, Elongin C, Cullin-2, RBX1)

  • Study how mutations affect complex formation through Western blot analysis

  • Investigate the BC-box motif's role in mediating these protein-protein interactions

The antibody's specificity for human VHL makes it particularly suitable for studying the molecular mechanisms underlying VHL disease and hypoxia response pathways in human cell models.

What approaches can be used to study VHL mutations and their effect on protein function?

The VHL PAT82B10AT antibody can facilitate several experimental approaches to study VHL mutations:

• Expression analysis in patient-derived samples:

  • Compare VHL protein levels between normal and tumor tissues

  • Correlate protein expression with mutation status

  • Assess stability differences between wild-type and mutant proteins

• Domain-specific functional studies:

  • Compare recognition of wild-type vs. mutant VHL

  • Investigate mutations affecting the β-domain (approximately 100-residue NH2-terminal domain) versus the α-helical domain

  • Analyze how mutations impact the polar interface and linker regions

• Structure-function relationship studies:

  • Compare antibody reactivity with truncated or mutant VHL constructs

  • Investigate epitope accessibility in different structural conformations

  • Study how mutations affect VHL's ability to form the E3 ligase complex with Elongin B/C

What are common challenges when using VHL PAT82B10AT antibody and how can they be addressed?

When optimizing and troubleshooting, remember that each investigation requires titration of the reagent to obtain optimal results for the specific application and experimental system .

How does sample preparation affect VHL detection using the PAT82B10AT antibody?

Sample preparation significantly impacts successful VHL detection:

• Cell/tissue lysis considerations:

  • Use RIPA or NP-40 based buffers for most applications

  • Include protease inhibitors to prevent VHL degradation

  • Perform lysis at 4°C to minimize proteolysis

  • Consider phosphatase inhibitors if studying VHL phosphorylation

• For Western blot sample preparation:

  • Denature samples thoroughly (95°C for 5 minutes) to expose the epitope

  • Use fresh reducing agents (β-mercaptoethanol or DTT)

  • Optimize protein loading (typically 20-50 μg total protein)

• For immunofluorescence:

  • Formalin fixation has been validated (as used for Daudi cells)

  • Optimize permeabilization conditions to access intracellular epitopes

  • Consider non-ionic detergents (0.1-0.5% Triton X-100 or 0.1% Tween-20)

• For capturing protein complexes:

  • Milder lysis conditions may preserve protein-protein interactions

  • Crosslinking prior to lysis can stabilize transient interactions

  • Consider native PAGE for analyzing intact complexes

How can the VHL PAT82B10AT antibody be integrated into studies of tumor hypoxia and angiogenesis?

The VHL PAT82B10AT antibody can provide valuable insights into tumor hypoxia and angiogenesis mechanisms:

• Correlation studies in tumor models:

  • Examine VHL protein levels relative to hypoxic markers

  • Co-stain for VHL and angiogenic factors (VEGF, PDGF)

  • Compare VHL expression patterns between normoxic and hypoxic regions

• Mechanistic investigations:

  • Monitor VHL-mediated regulation of HIF-1α stability under varying oxygen tensions

  • Assess how VHL status affects downstream angiogenic pathways

  • Study the dynamics of VHL-HIF interactions during hypoxic response

• Therapeutic response monitoring:

  • Evaluate how anti-angiogenic therapies affect VHL expression and function

  • Assess VHL status as a potential biomarker for treatment response

  • Investigate combination approaches targeting both VHL and its downstream effectors

Since VHL plays a crucial role as the substrate recognition component that ubiquitinates proteins with hydroxyproline residues, including HIF1α, this antibody enables researchers to study the molecular mechanisms connecting oxygen sensing to angiogenic response in cancer models .

What insights can studying VHL provide for understanding E3 ubiquitin ligase complexes?

VHL research using the PAT82B10AT antibody offers valuable insights into E3 ubiquitin ligase biology:

• Complex assembly and architecture:

  • Investigate how VHL interacts with Elongin B/C through the "BC-box motif"

  • Study how this heterodimeric complex connects substrate recognition to the E3 ligase machinery

  • Examine the role of VHL in recruiting Cullin-2/RBX1 to form the complete E3 ligase

• Substrate recognition mechanisms:

  • Analyze how VHL specifically recognizes hydroxyproline-containing substrates

  • Compare binding affinities between different VHL targets (HIF1α, β2-adrenergic receptor, ZHX2)

  • Investigate structural requirements for efficient substrate ubiquitination

• Regulatory mechanisms:

  • Study how post-translational modifications of VHL affect its function

  • Examine environmental conditions that modulate VHL activity

  • Investigate potential feedback mechanisms in the VHL-E3 ligase system

Understanding VHL's function provides a model for studying other substrate recognition components in E3 ligase complexes, particularly those in the VHL-box and SOCS-box protein families that utilize similar molecular mechanisms .