SENP5 Antibody

What is SENP5 and what are its primary cellular functions?

SENP5 belongs to the peptidase C48 family and functions as a SUMO-specific protease with dual catalytic activities in the SUMO pathway. It processes full-length SUMO3 to its mature form and deconjugates SUMO2 and SUMO3 from targeted proteins . SENP5 is essential for cell division, with knockdown studies demonstrating its requirement for proper mitosis and cytokinesis. Cells with reduced SENP5 expression show inhibited proliferation, defective nuclear morphology, and binucleate cell formation .

A particularly interesting aspect of SENP5 biology is its dynamic subcellular localization. During interphase, SENP5 predominantly localizes to the nucleolus, but translocates to mitochondria during the G2/M transition prior to nuclear envelope breakdown . This translocation coincides with changes in mitochondrial SUMOylation and increased mitochondrial fragmentation, suggesting SENP5 plays a key role in interorganellar communication during cell cycle progression .

What applications can SENP5 antibodies be used for in research?

Based on validation data from commercial suppliers, SENP5 antibodies have been successfully employed in multiple research applications:

When selecting a SENP5 antibody, researchers should consider the specific applications required for their studies and review validation data available from manufacturers to ensure suitability for their experimental systems .

What molecular weight should I expect to observe for SENP5 in Western blot experiments?

When performing Western blot analysis for SENP5, researchers should expect to observe a band at approximately 97 kDa, although the calculated molecular weight is 87 kDa . This discrepancy between calculated and observed molecular weights is not uncommon for proteins and may result from post-translational modifications or structural features affecting migration in SDS-PAGE.

It's worth noting that multiple SENP5 isoforms have been described with sizes ranging from 163-755 amino acids . When using antibodies targeting specific regions of SENP5, researchers should consider which isoforms contain the epitope recognized by their antibody. For example, the Novus antibody NB100-56412 was developed against a synthetic peptide corresponding to amino acids 229-246 of human SENP5 . Researchers are encouraged to BLAST the sequence to determine which isoforms would be recognized .

How does SENP5 discriminate between different SUMO family proteins, and how can antibodies help investigate this specificity?

SENP5 exhibits differential activity toward SUMO family proteins. In vitro studies demonstrate that SENP5's catalytic domain shows higher efficiency for processing SUMO-3 over SUMO-1 precursors. Similarly, SENP5 more efficiently removes SUMO-2 and SUMO-3 than SUMO-1 from SUMO-modified RanGAP1 . This contrasts with the activities described for other SUMO proteases like SENP1 and SENP2 .

Interestingly, in vivo, SENP5 displays isopeptidase activity toward both SUMO-1 and SUMO-2 conjugates, though with enhanced activity on SUMO-2 conjugates . This suggests complex regulatory mechanisms determine SENP5 substrate specificity in cellular contexts.

Researchers investigating this specificity can employ SENP5 antibodies in combination with antibodies against different SUMO paralogs in co-immunoprecipitation experiments to capture and analyze SENP5-substrate complexes. Alternatively, a proximity ligation assay could be used to visualize interactions between SENP5 and different SUMO-modified proteins in situ, providing spatial information about where these interactions occur within cells.

What role does SENP5 translocation from nucleoli to mitochondria play in cell cycle progression, and how can this be experimentally tracked?

The translocation of SENP5 from nucleoli to mitochondria during the G2/M transition is a significant cellular event with important implications for mitochondrial dynamics and cell cycle progression. This translocation results in reduced mitochondrial SUMOylation and increases the labile pool of DRP1 that drives mitochondrial fragmentation . Importantly, silencing SENP5 leads to cell cycle arrest precisely when the protease would normally translocate to mitochondria, suggesting this movement is critical for cell cycle progression .

To experimentally track this translocation, researchers could employ several approaches:

• Live-cell imaging using GFP-tagged SENP5 combined with mitochondrial and nucleolar markers

• Immunofluorescence of endogenous SENP5 at different cell cycle stages

• Biochemical fractionation to isolate cytosolic, nuclear, nucleolar, and mitochondrial fractions followed by Western blotting for SENP5

• Proximity labeling approaches (BioID or APEX) with SENP5 as bait to identify dynamic interaction partners during translocation

Data from fractionation experiments demonstrate a clear shift of endogenous SENP5 to mitochondrial fractions during specific cell cycle phases . Combining these approaches with cell synchronization techniques or live cell cycle sensors would provide comprehensive insights into the dynamics and functional significance of SENP5 translocation.

How does the non-catalytic N-terminal domain of SENP5 contribute to its localization and function, and what antibody-based approaches can reveal this relationship?

The SENP5 protein has distinct functional domains with the non-catalytic N-terminal region playing crucial roles in determining subcellular localization and potentially modulating catalytic activity. Research has shown that deletion of this N-terminal domain leads to two significant changes: loss of nucleolar localization and increased de-SUMOylation activity in vivo .

This finding suggests the N-terminal domain serves as both a localization signal and potentially a regulatory element that may restrict SENP5's catalytic activity until properly localized or activated. This dual functionality makes the N-terminal domain an interesting target for structure-function studies.

Researchers can investigate this relationship using domain-specific antibodies or epitope-tagged truncation constructs. Possible experimental approaches include:

• Generating domain-specific antibodies that recognize either the N-terminal or catalytic domains

• Using these antibodies to track the localization of endogenous SENP5 fragments after potential proteolytic processing

• Performing chromatin immunoprecipitation (ChIP) with N-terminal-specific antibodies to identify potential nucleolar binding sites

• Conducting activity assays with immunoprecipitated full-length versus truncated SENP5 to directly measure how the N-terminal domain affects catalytic efficiency

These approaches would provide insights into how structural elements of SENP5 coordinate its localization and enzymatic activity within cellular contexts.

What are the optimal fixation and antigen retrieval methods for SENP5 immunohistochemistry?

Successful immunohistochemical detection of SENP5 requires careful consideration of fixation and antigen retrieval methods. Based on validated protocols, researchers have achieved positive IHC detection in human cervical cancer tissue and human placenta tissue using the following approach :

The primary recommended antigen retrieval method is:

• TE buffer at pH 9.0

Alternatively, if this method doesn't yield optimal results:

• Citrate buffer at pH 6.0 can be used as an alternative

The choice between these methods may depend on tissue type, fixation duration, and the specific epitope recognized by the antibody. Researchers are advised to optimize these conditions for their particular tissue samples, as overfixation can mask epitopes and insufficient retrieval can lead to weak or absent staining.

What controls should be included when validating SENP5 antibody specificity in knockdown/knockout experiments?

When using SENP5 antibodies to validate knockdown or knockout experiments, proper controls are essential to ensure specificity and reliability of results. Based on published methodologies, researchers should consider the following controls:

• Positive controls:

  • Cell lines known to express SENP5 (HeLa cells have been validated)

  • Overexpression of tagged SENP5 constructs (3×Flag-SENP5 has been used)

• Negative controls:

  • Scrambled siRNA (e.g., scrambled version of Sp3 siRNA has been employed)

  • Vector-only controls when using shRNA approaches

• Validation approach:

  • When validating knockdown efficiency, researchers have successfully used co-transfection of SENP5 RNAis with 3×Flag-SENP5 (at a ratio of 10:1)

  • Nuclear extracts prepared 72h post-transfection can be analyzed using anti-Flag M2 (1:1,000) and anti-GFP (1:400) as loading control

• Multiple siRNA sequences:

  • To control for off-target effects, use multiple siRNA sequences targeting different regions of SENP5 mRNA

  • Published sequences include SENP5 RNAi 133 (5′-GAA AGC TAA GCT GGG AAG GCA-3′) and SENP5 RNAi 1111 (5′-GG GAG TGT ACA GAG CTG ATT A-3′)

• Phenotypic validation:

  • Confirm knockdown effects through functional assays such as cell cycle analysis

  • Published protocols have used flow cytometry with propidium iodide staining (50 μg/ml) and RNase A (100 μg/ml) treatment for 20 minutes at room temperature

What antibody dilutions and detection methods are optimal for different SENP5 experimental applications?

Optimizing antibody dilutions and detection methods is crucial for successful experiments across different applications. Based on validated protocols, here are recommended parameters for SENP5 antibodies:

For co-localization studies in immunofluorescence experiments, a validated approach includes:

• Fixation with methanol-acetone (1:1) for 20 min at −20°C

• Blocking with a serum mixture containing donkey serum (1:10), 0.1% Tween 20, 0.1% Triton X-100, and 3% bovine serum albumin

• Primary antibody incubation at room temperature for 1h or at 4°C overnight

• Secondary detection using fluorophore-conjugated antibodies such as Cy3-conjugated AffiniPure donkey anti-rabbit IgG (1:2,000)

What are potential explanations for SENP5 antibody failure to detect translocation between cellular compartments?

Failure to detect SENP5 translocation between cellular compartments (particularly from nucleoli to mitochondria during cell cycle progression) could result from several technical or biological factors:

• Cell cycle synchronization issues:

  • SENP5 translocation occurs specifically during G2/M transition

  • Unsynchronized cell populations may show predominantly nucleolar localization, masking the smaller fraction of cells with mitochondrial SENP5

  • Solution: Use cell synchronization methods such as double thymidine block or nocodazole treatment to enrich for G2/M phase cells

• Fixation artifacts:

  • Harsh fixation may disrupt the delicate balance of SENP5 localization

  • Solution: Compare multiple fixation methods (paraformaldehyde, methanol-acetone) to determine optimal preservation of subcellular structures

• Antibody epitope masking:

  • The epitope recognized by the antibody may become masked during translocation due to conformational changes or protein-protein interactions

  • Solution: Use antibodies targeting different epitopes or employ epitope-tagged SENP5 constructs

• Insufficient fractionation purity:

  • Cross-contamination between cellular fractions may obscure translocation events

  • Solution: Validate fractionation quality using compartment-specific markers (nucleolin for nucleoli, cytochrome c for mitochondria)

• Post-translational modifications:

  • Modifications may alter antibody recognition in different cellular compartments

  • Solution: Use multiple antibodies recognizing different epitopes or employ tagged SENP5 constructs

Researchers investigating SENP5 translocation should consider employing multiple detection methods, including live-cell imaging with fluorescently-tagged SENP5, immunofluorescence of endogenous protein, and biochemical fractionation followed by Western blotting .