SIRT2 Antibody, HRP conjugated

What is the optimal dilution range for SIRT2 antibody, HRP conjugated in Western blot applications?

The optimal dilution for SIRT2 antibody, HRP conjugated typically ranges from 1:1000 to 1:5000 for Western blot applications, depending on the specific antibody and sample concentration. For maximum sensitivity while maintaining low background, a titration experiment is recommended. Start with a dilution series (e.g., 1:1000, 1:2000, 1:5000, 1:10000) using positive control samples with known SIRT2 expression levels. The optimal dilution should provide clear detection of SIRT2 bands (typically observed at 37-45 kDa) with minimal background signal . Remember that SIRT2 exists in multiple isoforms (35 kDa, 40 kDa, and 42 kDa), so the banding pattern may vary based on the cell or tissue type and the epitope recognized by your specific antibody .

How should SIRT2 antibody, HRP conjugated be stored to maintain optimal activity?

SIRT2 antibody, HRP conjugated should typically be stored at -80°C for long-term storage to preserve enzymatic activity of the HRP conjugate and antibody stability . Avoid repeated freeze-thaw cycles as they can degrade both the antibody and the HRP enzyme. Upon first use, consider preparing small working aliquots stored at -20°C. When handling the antibody, keep it on ice and return to appropriate storage conditions promptly after use. Some formulations contain glycerol or stabilizing proteins that allow for 4°C storage of working aliquots for up to 2 weeks, but this varies by manufacturer and should be verified in the product documentation .

What blocking agents are most effective when using SIRT2 antibody, HRP conjugated for immunohistochemistry?

For immunohistochemistry applications using SIRT2 antibody, HRP conjugated, 5% normal serum (from the species in which the secondary antibody would normally be raised) or 3-5% BSA (bovine serum albumin) in PBS or TBS are typically most effective for blocking. Since HRP-conjugated antibodies eliminate the need for secondary antibodies, blocking should focus on reducing non-specific binding of the primary antibody. If background issues persist, consider:

• Including 0.1-0.3% Triton X-100 in the blocking solution for permeabilized sections

• Performing an additional blocking step with avidin/biotin if endogenous biotin is present

• Pre-incubation with 0.3% hydrogen peroxide to quench endogenous peroxidase activity

For paraffin-embedded tissues, proper antigen retrieval is crucial for optimal SIRT2 detection. Heat-induced epitope retrieval using citrate buffer (pH 6.0) has been shown to be effective for SIRT2 antibodies in multiple tissue types .

How can SIRT2 antibody, HRP conjugated be utilized in HTRF (Homogeneous Time-Resolved Fluorescence) assays for SIRT2 inhibitor screening?

SIRT2 antibody, HRP conjugated can be adapted for use in HTRF assays for inhibitor screening with careful consideration of assay design. Based on published protocols, HTRF screening for SIRT2 inhibitors typically involves:

• Modification of the standard HTRF format to incorporate the HRP-conjugated SIRT2 antibody instead of using a two-antibody system

• Optimization of signal-to-background ratio by adjusting antibody concentration (typically 0.1-0.3 nM final concentration)

• Implementation of appropriate controls to account for potential interference from the HRP moiety

The HTRF binding assay protocol can be adapted from published methods that use fluorescein-labeled substrate peptides. For example, a validated approach uses FAM-myristoyl-H4K16 peptide as a SIRT2 substrate with an apparent Kd of approximately 1 nM . When designing the assay, ensure that:

• The HRP conjugate doesn't interfere with the FRET pair signals

• The buffer conditions (10 mM Hepes-NaOH, pH 7.4, 150 mM NaCl, 5 mM DTT, and 0.005% Tween-20) are compatible with HRP activity

• The Z' factor is validated (should be >0.7 for a robust assay)

This adaptation allows for high-throughput screening of compound libraries to identify dual inhibitors of SIRT2's deacetylase and demyristoylase activities, particularly useful for drug discovery research.

How can non-specific background be reduced when using SIRT2 antibody, HRP conjugated in Western blots?

Non-specific background in Western blots using SIRT2 antibody, HRP conjugated can be minimized through several optimization strategies:

• Blocking optimization: Test different blocking agents (5% non-fat dry milk, 5% BSA, or commercial blocking buffers) to identify the optimal formulation. For SIRT2 detection, 5% BSA in TBST often provides superior results with reduced background.

• Antibody dilution adjustment: Increase the dilution of HRP-conjugated SIRT2 antibody (try 1:5000-1:10000) while extending the incubation time (overnight at 4°C) to maintain sensitivity while reducing background.

• Washing protocol enhancement:

  • Increase the number of wash steps (5-6 washes)

  • Extend wash duration (10 minutes per wash)

  • Use higher concentrations of Tween-20 in wash buffer (0.1-0.2%)

• Membrane optimization: PVDF membranes may provide better signal-to-noise ratio than nitrocellulose for SIRT2 detection. Pre-wet PVDF membranes in methanol followed by equilibration in transfer buffer.

If non-specific bands persist around the expected SIRT2 molecular weight (37-45 kDa), implement additional validation by including:

• SIRT2 knockout/knockdown controls

• Peptide competition assay using the immunizing peptide

These approaches have been demonstrated to effectively reduce background while maintaining specific detection of SIRT2 isoforms across multiple tissue and cell types .

What strategies can address inconsistent SIRT2 detection patterns across different tissue samples?

Inconsistent SIRT2 detection patterns across tissue samples may stem from several factors that can be systematically addressed:

• Isoform expression variations: SIRT2 exists in multiple isoforms (35-42 kDa), with tissue-specific expression patterns. Create a tissue-specific reference table documenting expected banding patterns for your antibody across tissues of interest .

• Epitope accessibility differences: Different fixation methods and tissue processing can affect epitope exposure. Implement a standardized fixation protocol and validate various antigen retrieval methods for each tissue type:

• Cross-reactivity considerations: Some SIRT2 antibodies may cross-react with other sirtuin family members. Validate specificity using:

  • Peptide competition assays

  • Tissues from SIRT2 knockout models

  • Parallel testing with multiple SIRT2 antibodies targeting different epitopes

• Sample preparation standardization: Develop tissue-specific lysate preparation protocols that optimize SIRT2 extraction and preserve post-translational modifications. For tissues with high lipid content (brain, liver), include additional detergent (0.5% NP-40) in lysis buffers .

By systematically addressing these factors, researchers can develop tissue-specific protocols that yield consistent SIRT2 detection patterns across experiments.

How can SIRT2 antibody signal be distinguished from potential cross-reactivity with other sirtuin family members?

Distinguishing SIRT2 signal from potential cross-reactivity with other sirtuin family members requires implementation of several validation strategies:

• Sequence alignment analysis: Review the immunogen sequence of your SIRT2 antibody (often a 19-amino acid synthetic peptide near the C-terminus ) and perform sequence alignment with other sirtuin family members to identify potential cross-reactive regions.

• Validation with recombinant proteins: Create a validation panel using:

  • Purified recombinant SIRT1, SIRT2, SIRT3, and SIRT7 proteins

  • Run Western blot with consistent protein amounts

  • Compare detection patterns and signal intensity

• Genetic knockout/knockdown controls: Implement:

  • SIRT2 siRNA or shRNA knockdown

  • CRISPR/Cas9 SIRT2 knockout cells

  • These should show significant reduction in the specific SIRT2 band

• Peptide competition assay: Pre-incubate SIRT2 antibody with:

  • SIRT2 immunizing peptide (should abolish signal)

  • Peptides from homologous regions of other sirtuins (should not affect SIRT2-specific signal)

• Dual detection strategy: Use two SIRT2 antibodies targeting different epitopes in sequential or parallel detection to confirm specificity.

A common pattern of cross-reactivity is between SIRT2 and SIRT3 due to sequence homology. When detecting SIRT2 in mitochondria-rich tissues (heart, liver), always include appropriate controls to distinguish between these two sirtuins, as SIRT3 is predominantly mitochondrial, while SIRT2 is primarily cytosolic with some nuclear localization .

What are the critical parameters for quantifying SIRT2 enzymatic activity using SIRT2 antibody, HRP conjugated in an ELISA-based deacetylase assay?

Developing an ELISA-based deacetylase assay using SIRT2 antibody, HRP conjugated requires careful optimization of several critical parameters:

• Substrate selection and immobilization:

  • Use acetylated peptide substrates (e.g., acetylated H4K16 peptides) immobilized on ELISA plates

  • Ensure consistent coating density (typically 1-5 μg/ml peptide)

  • Block non-specific binding sites thoroughly with 3% BSA

• Enzymatic reaction conditions:

  • Purified SIRT2 protein concentration: 50-200 ng/well

  • NAD+ concentration: 0.5-1 mM (critical co-factor)

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂

  • Reaction time: 30-60 minutes at 37°C

  • Include controls: without NAD+, with SIRT2 inhibitor (e.g., AGK2 at 10 μM)

• Detection parameters:

  • Use acetyl-lysine specific antibody followed by SIRT2 antibody, HRP conjugated

  • Alternatively, use SIRT2 antibody, HRP conjugated to detect SIRT2 bound to substrate

  • Optimize antibody concentration (typically 1:1000-1:5000)

  • Select appropriate HRP substrate for detection (TMB provides high sensitivity)

• Data analysis considerations:

  • Calculate percent deacetylation relative to control wells

  • Use non-linear regression for enzyme kinetics analysis

  • Apply Michaelis-Menten equation to determine Km and Vmax

The table below shows typical optimization parameters for the assay:

This approach allows for quantitative assessment of SIRT2 deacetylase activity, suitable for inhibitor screening and comparative analysis of SIRT2 activity across different experimental conditions .

What controls are essential when using SIRT2 antibody, HRP conjugated to study SIRT2's role in neurodegenerative diseases?

When investigating SIRT2's role in neurodegenerative diseases, several essential controls must be incorporated:

• Tissue-specific expression controls:

  • Age-matched control tissues from non-diseased subjects

  • Brain region-matched controls (SIRT2 is highly expressed in brain but varies by region)

  • Cell type-specific controls (neurons vs. glia, as SIRT2 expression differs)

• Disease model validation controls:

  • Established disease markers (e.g., α-synuclein aggregates for Parkinson's disease)

  • Time-course analysis in progressive models

  • Pharmacological validation using known disease modulators

• Antibody specificity controls:

  • SIRT2 knockout/knockdown neural cells

  • Peptide competition assays specific to neural tissue

  • Cross-validation with multiple SIRT2 antibodies recognizing different epitopes

• Functional correlation controls:

  • Parallel assessment of known SIRT2 substrates (α-tubulin acetylation)

  • SIRT2 activity assays in neural lysates

  • SIRT2 inhibitor treatment (e.g., AGK2) to confirm causality

• Comprehensive control panel for immunohistochemistry:

These controls are particularly important in neurodegenerative disease research where SIRT2 has been implicated in multiple pathways, including α-synuclein aggregation, microtubule stability, and oxidative stress response .

What methodology would you recommend for using SIRT2 antibody, HRP conjugated to study post-translational modifications of SIRT2 itself?

Studying post-translational modifications (PTMs) of SIRT2 using SIRT2 antibody, HRP conjugated requires a sophisticated methodological approach:

• Two-dimensional Western blot analysis:

  • First dimension: Isoelectric focusing to separate SIRT2 based on charge differences from PTMs

  • Second dimension: SDS-PAGE separation by molecular weight

  • Detection: SIRT2 antibody, HRP conjugated

  • This approach reveals SIRT2 "spots" representing differently modified forms

• PTM-specific antibody sequential detection:

  • Immunoprecipitate SIRT2 using non-HRP conjugated antibodies

  • First detection: PTM-specific antibodies (phospho, acetyl, SUMO, ubiquitin)

  • Strip membranes and re-probe with SIRT2 antibody, HRP conjugated

  • Calculate modified:total SIRT2 ratios

• Mass spectrometry workflow:

  • Immunoprecipitate SIRT2 from cells under various conditions

  • Perform tryptic digestion and LC-MS/MS analysis

  • Confirm identified modifications by targeted mass spectrometry

  • Validate findings using site-specific antibodies or site-directed mutagenesis

• PTM-specific functional correlation:

  • Generate phosphomimetic or phospho-deficient SIRT2 mutants

  • Assess impact on:

    • Localization (phosphorylation often affects nuclear localization)

    • Enzymatic activity (using deacetylase assays)

    • Substrate specificity (using deacetylase assays with different substrates)

    • Protein-protein interactions (using co-immunoprecipitation)

• Recommended validation protocol:

This comprehensive approach enables detailed characterization of how SIRT2 itself is regulated through various PTMs, providing insight into the complex regulatory mechanisms controlling SIRT2 function in different cellular contexts .

What emerging applications of SIRT2 antibody, HRP conjugated should researchers consider for future studies?

Emerging applications of SIRT2 antibody, HRP conjugated that researchers should consider include:

• Single-cell proteomics applications:

  • Adaptation for mass cytometry (CyTOF) through metal-conjugated SIRT2 antibodies

  • Integration with spatial transcriptomics to correlate SIRT2 protein levels with gene expression patterns

  • Microfluidic-based single-cell Western blotting for heterogeneity analysis

• Extracellular vesicle (EV) SIRT2 detection:

  • Analyzing SIRT2 packaging into exosomes and microvesicles

  • Developing sensitive ELISA-based detection methods for SIRT2 in isolated EVs

  • Correlating circulating EV SIRT2 levels with disease states

• Advances in super-resolution microscopy:

  • Implementing STORM or PALM imaging with converted SIRT2 antibodies

  • Investigating nanoscale co-localization with cytoskeletal elements

  • Analyzing SIRT2 clustering dynamics during cell cycle progression

• Drug screening and therapeutic development:

  • High-content phenotypic screening using SIRT2 antibodies to identify modulators

  • Target engagement assays to confirm drug binding to SIRT2 in intact cells

  • Companion diagnostic development for SIRT2-targeted therapeutics

These applications will provide deeper insights into SIRT2's multifaceted roles in cellular function and disease processes, particularly in cancer, neurodegeneration, and metabolic disorders. The continued development of highly specific and sensitive SIRT2 detection methods will be crucial for advancing our understanding of this important regulatory protein .

How might contradictory findings regarding SIRT2's role in disease progression be reconciled through improved antibody-based methodologies?

Contradictory findings regarding SIRT2's role in disease progression can be reconciled through several improved antibody-based methodological approaches:

• Isoform-specific detection and functional analysis:

  • Develop and validate isoform-specific SIRT2 antibodies

  • Implement parallel detection of all SIRT2 isoforms in the same samples

  • Correlate isoform expression patterns with disease progression

• Context-dependent activity assessment:

  • Develop in situ activity-based probes for SIRT2

  • Combine with traditional antibody detection

  • Map activity:expression ratios across tissue sections

• Integrated multi-omics approach:

  • Correlate antibody-based SIRT2 protein detection with:

    • PTM status (phosphoproteomics)

    • Interactome analysis (BioID or proximity labeling)

    • Metabolomics (NAD+ availability)

    • Acetylome profiling (substrate modification status)

• Tissue and cell type heterogeneity resolution:

  • Single-cell proteomic analysis of SIRT2 levels

  • Spatial proteomics to map SIRT2 distribution within tissues

  • Cell type-specific SIRT2 knockout/knockin models

• Temporal dynamics consideration:

  • Time-course studies with standardized antibody-based detection

  • Pulse-chase experiments to assess SIRT2 protein turnover

  • Live-cell imaging of SIRT2 dynamics

These approaches would help resolve contradictions that often arise from failing to account for SIRT2 isoform differences, cell type-specific functions, temporal dynamics of SIRT2 activity, or context-dependent regulation of SIRT2. By implementing these methodological improvements, researchers can develop a more nuanced understanding of SIRT2's complex and sometimes opposing roles in different disease contexts .

What methodological considerations should guide researchers in selecting between different SIRT2 antibody formats for specialized research applications?

When selecting between different SIRT2 antibody formats for specialized research applications, researchers should consider these methodological factors:

• Application-specific performance characteristics:

• Epitope accessibility considerations:

  • N-terminal epitopes: Better for detecting all SIRT2 isoforms

  • C-terminal epitopes: May be masked in certain protein complexes

  • Internal epitopes: May be affected by conformational changes

• Signal amplification requirements:

  • Direct HRP conjugates: Suitable for abundant targets

  • Biotin-conjugated + streptavidin-HRP: Enhanced sensitivity for low-abundance detection

  • Tyramide signal amplification: Maximum sensitivity for trace detection

• Multiplexing capabilities:

  • HRP-conjugated: Limited to sequential detection with stripping

  • Fluorophore-conjugated: Enables simultaneous multi-target detection

  • Unconjugated primary + species-specific secondary: Maximum flexibility

• Buffer and fixation compatibility:

  • Formaldehyde-sensitive epitopes: Select antibodies validated for IHC/ICC

  • Detergent sensitivity: Some conjugates may have reduced activity in high-detergent buffers

  • pH sensitivity: Confirm stability across buffer conditions needed for your application