SENP6 Antibody, HRP conjugated

What is SENP6 and why is it significant in cellular research?

SENP6 is a protease that specifically deconjugates SUMO1, SUMO2, and SUMO3 from targeted proteins, with a preference for processing poly-SUMO2 and poly-SUMO3 chains. It is significant in cellular research because it regulates multiple crucial cellular processes including chromosome alignment, spindle assembly, DNA repair, and transcriptional activation . SENP6 functions by regulating the kinetochore CENPH-CENPI-CENPK complex and protecting proteins like PML and CENPI from degradation by the ubiquitin ligase RNF4 . Additionally, SENP6 has been identified as a tumor suppressor in lymphomas, making it a potential target for cancer research .

What applications are SENP6 antibodies suitable for in research settings?

SENP6 antibodies have demonstrated efficacy in multiple research applications:

• Western Blotting (WB): For detecting SENP6 protein expression levels

• Immunoprecipitation (IP): For isolating SENP6 and its binding partners

• Immunofluorescence (IF): For visualizing cellular localization of SENP6

• Immunohistochemistry with paraffin-embedded sections (IHC-P): For tissue-level expression analysis

• Enzyme-linked immunosorbent assay (ELISA): For quantitative measurement of SENP6

The specific applications vary between antibodies, with some optimized for particular techniques. For example, the SENP6 Antibody (79-M) from Santa Cruz detects human SENP6 by WB, IP, IF, IHC(P), and ELISA , while some rabbit polyclonal antibodies may have more limited application ranges .

What is the difference between HRP-conjugated SENP6 antibodies and non-conjugated versions?

HRP (Horseradish Peroxidase)-conjugated SENP6 antibodies have the enzyme directly attached to the antibody molecule, eliminating the need for secondary antibody incubation in certain applications. This provides several advantages:

• Simplified workflow: Reduces experimental time by eliminating secondary antibody incubation steps

• Reduced background: Minimizes non-specific binding that can occur with secondary antibodies

• Enhanced sensitivity: Often provides stronger signal with less primary antibody required

• Direct detection: Allows for immediate visualization upon addition of substrate

Non-conjugated antibodies require a secondary antibody step but offer greater flexibility in detection methods and signal amplification strategies. The choice between conjugated and non-conjugated depends on your specific experimental needs, with HRP-conjugated being particularly advantageous for ELISA and certain Western blotting protocols .

How should SENP6 antibodies be stored to maintain optimal activity?

For optimal maintenance of SENP6 antibody activity, proper storage conditions are essential:

• Temperature: Store at -20°C for long-term storage or at 4°C for short-term use

• Buffer conditions: Most SENP6 antibodies are supplied in buffers containing preservatives and stabilizers

• For HRP-conjugated SENP6 antibodies specifically: Store in 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as a preservative

• Avoid repeated freeze-thaw cycles: Aliquot antibodies upon initial thawing to prevent degradation

• Protect from light: Particularly important for fluorophore-conjugated antibodies

Following these storage guidelines will help maintain antibody activity and ensure consistent experimental results over time.

How does SENP6 knockdown affect centromere protein dynamics and what methods can detect these changes?

SENP6 knockdown significantly impacts centromere protein dynamics, as revealed through advanced imaging and biochemical studies. Following SENP6 depletion:

• Accumulation of CENP-T, CENP-W, and CENP-A to centromeres is markedly impaired

• Massive buildup of SUMO chains occurs on CENP-B, -C, -H, -I, -K, and -T, extending to high molecular weights on protein gels

• SUMOylated CCAN subunits show reduced abundance at chromatin and centromeres

To detect these changes, researchers can employ:

• Immunofluorescence microscopy with anti-CENP antibodies to visualize centromere localization

• Western blotting with SENP6 antibodies followed by reprobing with CENP antibodies to observe SUMOylation patterns

• Chromatin immunoprecipitation assays to quantify CCAN subunit association with centromeres

• Cell cycle analysis to detect G2/M accumulation and micronuclei formation that result from compromised centromere function

Interestingly, contrary to the classic understanding of poly-SUMO2/3 accumulation leading to proteasomal degradation, CCAN proteins do not show increased degradation despite poly-SUMOylation, suggesting a proteolysis-independent mechanism of SUMO polymer signaling .

What is the relationship between SENP6 deficiency and DNA damage response, and how can SENP6 antibodies help investigate this connection?

SENP6 deficiency significantly compromises DNA damage response mechanisms, creating genomic instability that has implications for both normal cellular function and cancer development. This relationship manifests through several mechanisms:

• SENP6 loss triggers the release of DNA repair and genome maintenance-associated protein complexes from chromatin

• SENP6 desumoylates RPA1, preventing RAD51 recruitment to DNA damage foci, which impairs homologous recombination repair

• SENP6 deficiency leads to synthetic lethality with PARP inhibition, indicating its role in alternative DNA repair pathways

SENP6 antibodies can help investigate this connection through:

• Chromatin fractionation experiments followed by Western blotting to assess DNA repair protein displacement from chromatin

• Co-immunoprecipitation with SENP6 antibodies to identify interactions with DNA repair factors

• Immunofluorescence to visualize SENP6 colocalization with DNA damage markers like γH2AX

• ChIP-seq analysis after DNA damage to map SENP6 recruitment to damage sites

This research direction has therapeutic implications, as SENP6-deficient lymphomas showed increased sensitivity to PARP inhibitors, suggesting a potential precision medicine approach for patients with SENP6 alterations .

How do SUMO chain dynamics change after SENP6 knockdown and what technical approaches can quantify these changes?

Following SENP6 knockdown, SUMO chain dynamics undergo dramatic alterations that can be quantified through various technical approaches:

• SUMO Chain Accumulation Patterns:

  • Massive buildup of SUMO chains on target proteins with conjugates extending to very high molecular weights

  • Preferential accumulation of poly-SUMO2 and poly-SUMO3 chains compared to SUMO1

  • Highly interconnected networks of proteins become hyperSUMOylated, demonstrating group deSUMOylation by SENP6

• Quantification Approaches:

  • Proteomic analysis using SUMO-IP followed by mass spectrometry to identify enriched proteins (showed up to 900-fold enrichment of some targets after SENP6 knockdown)

  • Sequential immunoprecipitation of SUMO and ubiquitin to measure changes in dual-modified proteins

  • Western blotting with anti-SUMO antibodies to visualize the characteristic ladder pattern of poly-SUMOylation

  • ELISA-based quantification of global SUMOylation levels

• Unexpected Dynamics:

  • Proteasome inhibition paradoxically reduces SUMOylation of CCAN family members (CENP-K, -T, -I, -C, and -H) rather than increasing it, contrary to the expected stabilization of SUMOylated species

  • This suggests a proteolysis-independent function of SUMO chains in regulating these proteins

These approaches collectively enable researchers to dissect the complex regulatory networks controlled by SENP6-mediated SUMO chain processing.

What is the significance of SENP6 in cancer research and how can SENP6 expression levels be effectively analyzed in tumor samples?

SENP6 has emerged as a significant tumor suppressor, particularly in B-cell lymphomas, with important implications for cancer research:

• Tumor Suppressor Role:

  • SENP6 is recurrently deleted in human lymphomas

  • Transposon mutagenesis screens identified SENP6 as a tumor suppressor in MYC-driven B-cell lymphoma models

  • Low SENP6 expression correlates with primary refractory disease or early relapse in DLBCL patients (P = 0.0211)

• Effective Analysis Methods for SENP6 Expression:

  • Immunohistochemistry on tissue microarrays (TMAs) to categorize tumors as SENP6-high vs. SENP6-low based on nuclear staining

  • Western blotting with validated SENP6 antibodies for protein level quantification

  • RT-qPCR for mRNA expression analysis

  • FISH (Fluorescent In Situ Hybridization) to detect SENP6 gene deletion

• Therapeutic Implications:

  • SENP6 deficiency creates a synthetic lethal interaction with PARP inhibitors, suggesting potential targeted therapy approaches

  • SU-DHL-5 DLBCL cells show increased sensitivity to doxorubicin treatment after SENP6 expression reconstitution

This research direction offers promising avenues for both biomarker development and precision medicine approaches in lymphoma treatment.

What are the optimal conditions for using HRP-conjugated SENP6 antibodies in Western blotting experiments?

For optimal results when using HRP-conjugated SENP6 antibodies in Western blotting, consider the following protocol recommendations:

• Sample Preparation:

  • Prepare protein lysates in RIPA buffer containing protease inhibitors

  • Include SUMO protease inhibitors (like N-ethylmaleimide, 20mM) to preserve SUMOylation status

  • Use fresh samples when possible as freeze-thaw cycles can affect SUMOylation patterns

• Gel and Transfer Parameters:

  • Use gradient gels (4-12% or 4-20%) to effectively resolve both unmodified SENP6 (~126 kDa) and SUMOylated species

  • Extend running time to properly separate high molecular weight SUMO conjugates

  • Use PVDF membrane for transfer as it provides better protein retention than nitrocellulose

• Antibody Incubation:

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • For HRP-conjugated SENP6 antibodies, recommended dilutions range from 1:1000 to 1:2000

  • Incubate overnight at 4°C for maximum sensitivity

  • Wash thoroughly (5-6 times) with TBST to minimize background

• Detection:

  • Use enhanced chemiluminescence (ECL) substrates with extended signal duration

  • For detecting low abundance SUMOylated species, consider using super-sensitive ECL reagents

  • Imaging systems with broad dynamic range are preferable to film for quantitative analysis

• Controls:

  • Include a SENP6 knockdown sample as a negative control

  • Run a SENP6 overexpression sample as a positive control

  • Consider including proteasome inhibitor-treated samples to observe effects on SUMOylation patterns

Following these optimized conditions will help ensure reliable and reproducible detection of both native SENP6 and its SUMOylated targets.

How should experiments be designed to study the interaction between SENP6 and its target proteins?

To effectively study interactions between SENP6 and its target proteins, experimental design should incorporate multiple complementary approaches:

• Co-immunoprecipitation (Co-IP) Strategies:

  • Forward approach: Immunoprecipitate with anti-SENP6 antibodies and blot for suspected target proteins

  • Reverse approach: Immunoprecipitate target proteins (e.g., CENP-I, RPA1) and blot for SENP6

  • Cross-linking prior to IP can capture transient enzyme-substrate interactions

  • Include SUMO protease inhibitors in lysis buffers to preserve SUMOylated species

• Proximity Ligation Assays (PLA):

  • Allows visualization of protein-protein interactions in situ with single-molecule sensitivity

  • Particularly useful for detecting SENP6 interactions with low-abundance centromere proteins

  • Can be combined with cell synchronization to detect cell cycle-specific interactions

• CRISPR-Cas9 Approaches:

  • Generate SENP6 catalytic mutants to distinguish between binding and enzymatic activity

  • Create domain-specific mutations to map interaction surfaces

  • Employ SENP6 knockdown followed by rescue with mutant versions to assess functional consequences

• Proteomic Strategies:

  • SILAC or TMT labeling combined with SENP6 knockdown/overexpression to quantify changes in the SUMOylome

  • BioID or TurboID fusion proteins to identify proximity-based interactors of SENP6

  • Sequential purification of SUMO conjugates followed by mass spectrometry to identify SENP6 substrates

• Visualization Techniques:

  • Fluorescently-tagged SENP6 and target proteins for live cell imaging

  • FRET or BRET assays to measure direct interactions

  • Chromatin immunoprecipitation to identify co-occupancy at specific genomic loci

These approaches provide complementary data that together can establish both the physical interaction and functional consequences of SENP6-target relationships in various cellular contexts.

What controls should be included when using SENP6 antibodies in immunohistochemistry studies of tumor samples?

When conducting immunohistochemistry (IHC) studies of tumor samples with SENP6 antibodies, proper controls are essential for accurate interpretation and quantification:

• Essential Positive Controls:

  • Normal tissue with known SENP6 expression (e.g., testis or ovary tissues which exhibit high SENP6 expression)

  • Cell line pellets with verified SENP6 expression levels embedded alongside clinical samples

  • Internal positive controls within the sample (non-tumor cells with consistent SENP6 expression)

• Critical Negative Controls:

  • SENP6 knockout or knockdown cell lines processed identically to test samples

  • Primary antibody omission (substituted with antibody diluent) on serial sections

  • Isotype control antibody (IgG2a κ for mouse monoclonal or IgG for rabbit polyclonal) at the same concentration

  • Peptide competition assay using the immunizing peptide to confirm specificity

• Staining Pattern Validation:

  • Nuclear staining evaluation is critical as SENP6 functions primarily in the nucleus

  • Cytoplasmic staining should be evaluated with caution as some SENP6 is also present in cytoplasm

  • Compare patterns to published subcellular localization data

• Quantification Controls:

  • Include reference samples with established SENP6 expression levels (high/medium/low) in each batch

  • Use digital image analysis with validated algorithms when possible for objective scoring

  • Employ double-blind scoring by multiple pathologists to ensure reproducibility

• Technical Controls:

  • Process all samples using identical protocols (fixation time, antigen retrieval method, antibody concentration)

  • Include a tissue microarray with known SENP6 expression patterns across different tumors

  • Run parallel staining with different validated SENP6 antibody clones when possible

What are common issues when detecting SUMOylated targets of SENP6 and how can they be resolved?

Detecting SUMOylated targets of SENP6 presents several technical challenges due to the dynamic and often transient nature of SUMO modifications. Here are common issues and their solutions:

• Low Signal of SUMOylated Species:

  • Problem: SUMOylated targets often represent a small fraction of the total protein pool

  • Solution: Enrich SUMOylated proteins using SUMO immunoprecipitation before Western blotting

  • Solution: Include SUMO protease inhibitors (N-ethylmaleimide, 20mM) in all buffers

  • Solution: Use proteasome inhibitors cautiously, as they may unexpectedly decrease SUMOylation of some SENP6 targets like CCAN components

• Smeared or Ladder-like Bands:

  • Problem: Poly-SUMOylated proteins appear as high molecular weight smears rather than discrete bands

  • Solution: Use gradient gels (4-15%) with extended running times to better resolve high-molecular-weight species

  • Solution: Perform denaturing immunoprecipitation to reduce non-specific interactions

  • Solution: Consider using SUMO-2/3 specific antibodies as SENP6 preferentially processes these chains

• Inconsistent Results After SENP6 Knockdown:

  • Problem: Variable SUMOylation patterns in different experiments

  • Solution: Monitor knockdown efficiency by Western blotting for SENP6

  • Solution: Consider stable knockdown systems rather than transient transfection

  • Solution: Synchronize cells, as SUMOylation patterns vary throughout the cell cycle, particularly for centromere proteins

• Difficulty Distinguishing Direct vs. Indirect SENP6 Targets:

  • Problem: SENP6 knockdown affects global SUMOylation, making it difficult to identify direct substrates

  • Solution: Use catalytically inactive SENP6 mutants that bind but don't cleave substrates

  • Solution: Perform in vitro deSUMOylation assays with purified components

  • Solution: Compare SENP6 knockdown effects with other SENP family members to identify specific targets

• High Background in Immunofluorescence Studies:

  • Problem: Non-specific staining obscures SUMOylation signals at specific structures like centromeres

  • Solution: Pre-extract cells with detergent to remove soluble proteins before fixation

  • Solution: Use super-resolution microscopy techniques for better visualization of colocalization

  • Solution: Combine with proximity ligation assays to specifically detect SUMOylated target proteins

Implementing these troubleshooting approaches will significantly improve detection and characterization of SUMOylated SENP6 targets in various experimental contexts.

How can researchers address contradictory findings when studying SENP6 function in different cell types or experimental systems?

When confronting contradictory findings regarding SENP6 function across different experimental systems, researchers should implement a systematic approach to reconcile these discrepancies: