SIRT7 Antibody

What is SIRT7 and what cellular functions does it regulate?

SIRT7 (NAD-dependent deacetylase sirtuin-7), also known as SIR2L7, is a member of the class IV sirtuin family that functions as an NAD+-dependent protein deacetylase regulating cell growth and proliferation. SIRT7 is primarily localized to nucleoli, where it plays critical roles in RNA polymerase I (Pol I) transcription and pre-rRNA processing. Its expression correlates with cell growth, being high in metabolically active cells and low or absent in non-proliferating cells . In epithelial prostate carcinomas, high SIRT7 levels are associated with aggressive cancer phenotypes, metastatic disease, and poor patient prognosis .

SIRT7 regulates multiple cellular processes through its deacetylase activity, including:

• rDNA transcription via deacetylation of PAF53, a core subunit of mammalian Pol I

• Pre-rRNA processing through deacetylation of U3-55k, a component of the U3 snoRNP complex

• Transcription of snoRNAs and mRNAs

• Histone modification, particularly deacetylation of H3K18ac, a biomarker of aggressive tumors

SIRT7-knockout mice exhibit increased embryonic lethality, reduced stress resistance, inflammatory cardiomyopathy, and premature aging, demonstrating its importance in development and physiological homeostasis .

What is the molecular weight of SIRT7 and how is it detected in western blots?

SIRT7 has a calculated molecular weight of 45 kDa based on its 400 amino acid sequence, and this matches its observed migration pattern in SDS-PAGE gels . When performing western blots, SIRT7 consistently appears as a band at approximately 45 kDa across multiple cell lines and tissue samples.

For optimal western blot detection, the recommended dilution range is 1:1000-1:4000 for most SIRT7 antibodies . Positive controls with reliable SIRT7 expression include human cell lines (PC-3, HeLa, HEK-293T, HepG2, MCF7), mouse tissues (liver, kidney, spleen), and rat liver tissue . Standard western blot protocols with 20 μg of total protein are typically sufficient for detection .

Which species reactivity is most commonly available for SIRT7 antibodies?

Commercial SIRT7 antibodies typically demonstrate cross-reactivity with human, mouse, and rat samples . This multi-species reactivity is advantageous for comparative studies and translational research. Most validated SIRT7 antibodies are raised in rabbits, either as polyclonal (e.g., Proteintech 12994-1-AP) or monoclonal (e.g., Abcam ab259968) formulations .

Specific cell lines and tissues where SIRT7 antibody reactivity has been positively confirmed include:

Human cell lines:

• HeLa (cervix adenocarcinoma)

• HEK-293T (embryonic kidney)

• HepG2 (hepatocellular carcinoma)

• MCF7 (breast adenocarcinoma)

• PC-3 (prostate cancer)

Mouse tissues/cell lines:

• Liver and kidney tissues

• Hepa1-6 (hepatoma)

• NIH/3T3 (embryonic fibroblast)

• RAW264.7 (macrophage)

Rat tissues/cell lines:

• Liver tissue

• C6 (glial tumor cells)

What applications are SIRT7 antibodies typically used for?

SIRT7 antibodies have been validated for multiple experimental applications with specific recommended protocols for each:

According to published research data, Western blot is the most commonly used application (22+ publications), followed by IHC (5+ publications), with IP and IF each represented in at least one publication . SIRT7 antibodies have also been instrumental in knockdown/knockout validation studies, with at least 3 publications utilizing this approach .

How does SIRT7 localization change under cellular stress?

When cells experience stress, SIRT7 is released from nucleoli and accumulates in the nucleoplasm . This translocation has significant functional consequences:

• The redistribution leads to hyperacetylation of nucleolar SIRT7 substrates including PAF53 and U3-55k

• This hyperacetylation results in defects in both transcription and processing of pre-rRNA

• These changes contribute to stress-responsive downregulation of ribosome biogenesis

This shuttling mechanism represents a central regulatory process linking environmental stress signals to cellular growth control through modulation of ribosome biogenesis . The stress-induced redistribution of SIRT7 can be visualized using immunofluorescence techniques with specific SIRT7 antibodies.

How does SIRT7 regulate RNA polymerase II-mediated transcription?

Beyond its established role in ribosome biogenesis, SIRT7 plays a sophisticated role in regulating RNA polymerase II (Pol II)-mediated transcription through multiple interconnected mechanisms:

• P-TEFb regulation: SIRT7 promotes the release of P-TEFb (Positive Transcription Elongation Factor b) from the inactive 7SK snRNP complex. This is a crucial step in activating transcription elongation .

• CDK9 deacetylation: SIRT7 deacetylates CDK9, a key subunit of the P-TEFb complex. This deacetylation directly activates the kinase activity of CDK9 .

• CTD phosphorylation cascade: Activated CDK9 phosphorylates serine 2 within the C-terminal domain (CTD) of RNA polymerase II. This phosphorylation event is essential for productive transcription elongation .

• Transcriptional activation: Through these molecular events, SIRT7 facilitates transcription elongation of various Pol II-transcribed genes, including snoRNAs and mRNAs .

This regulatory pathway positions SIRT7 as a metabolic sensor (through NAD+ dependency) that links cellular energy status to gene expression programs. Proteomic analyses have revealed that SIRT7 associates with numerous proteins involved in transcriptional regulation and RNA metabolism, with many of these interactions requiring ongoing transcription .

What techniques are most effective for studying SIRT7-protein interactions?

Several complementary techniques can be employed to effectively characterize SIRT7's protein interaction network:

• Co-immunoprecipitation (Co-IP):

  • This is the most widely used method for studying SIRT7 interactions

  • Typically requires 0.35-1.0 mg of whole cell lysate with 1-4 μg of SIRT7 antibody

  • Protocol: Add 1 μg of SIRT7 antibody to cell lysate, incubate overnight at 4°C with rotation, then capture with protein A/G beads

  • Always include IgG control antibodies as negative controls

• Proteomic approaches:

  • Mass spectrometry following SIRT7 immunoprecipitation can identify novel interaction partners

  • Several studies have employed this approach to characterize the SIRT7 interactome, revealing interactions with proteins involved in transcription, ribosome biogenesis, and translation

• RNA-dependency studies:

  • Since many SIRT7 interactions depend on RNA, RNase treatment before immunoprecipitation can determine if interactions are direct or RNA-mediated

  • The N-terminal part of SIRT7 binds RNA and mediates RNA-dependent protein interactions

• Transcription-dependency studies:

  • Using transcription inhibitors before protein interaction studies can reveal which interactions require ongoing transcription

  • A large fraction of the SIRT7 interactome depends on active transcription

• Chromatin immunoprecipitation (ChIP):

  • For studying SIRT7 interactions with chromatin and DNA

  • Has revealed SIRT7 occupancy at specific genomic loci, including tRNA genes

How does SIRT7 deacetylation of CDK9 affect transcriptional elongation?

SIRT7's deacetylation of CDK9 represents a critical molecular switch in transcriptional regulation, affecting the expression of numerous genes. The mechanism involves several precisely orchestrated steps:

• Molecular mechanism:

  • CDK9 is a subunit of P-TEFb (Positive Transcription Elongation Factor b), a key factor controlling transcription elongation

  • SIRT7, utilizing NAD+ as a cofactor, removes acetyl groups from specific lysine residues on CDK9

  • This deacetylation directly activates CDK9's intrinsic kinase activity

• P-TEFb activation pathway:

  • SIRT7 promotes the release of P-TEFb from the inactive 7SK snRNP complex

  • The 7SK snRNP complex normally sequesters P-TEFb in an inactive state

  • By facilitating P-TEFb release and deacetylating CDK9, SIRT7 enables P-TEFb activation

• Pol II CTD phosphorylation:

  • Activated CDK9 phosphorylates serine 2 within the C-terminal domain (CTD) of RNA Polymerase II

  • This phosphorylation is the critical step for transitioning from transcription initiation to productive elongation

  • The phosphorylated CTD serves as a platform for recruiting factors involved in co-transcriptional processes

• Gene expression outcomes:

  • Enhanced transcription elongation of Pol II-transcribed genes

  • Affects transcription of both snoRNAs and mRNAs

  • Contributes to SIRT7's broader role in regulating cell growth and proliferation

This pathway demonstrates how SIRT7, through sensing cellular NAD+ levels, can coordinate gene expression with metabolic status, providing a mechanistic link between cellular metabolism and transcriptional control.

How does SIRT7 interact with chromatin remodeling complexes?

SIRT7 interacts with several chromatin remodeling complexes, contributing to its role in transcriptional regulation through chromatin structure modification:

• Interaction with specific complexes:

  • SIRT7 associates with multiple chromatin remodeling machineries including:

    • B-WICH complex

    • NoRC (Nucleolar Remodeling Complex)

    • SWI/SNF (Switch/Sucrose Non-Fermentable) complex

• Histone modification activities:

  • SIRT7 catalyzes deacetylation of lysine 18 at histone H3 (H3K18ac)

  • H3K18ac is a biomarker of aggressive tumors

  • Hypoacetylation of H3K18 compromises transcription of tumor suppressor genes and facilitates DNA repair

• Chromatin structure regulation:

  • These interactions collectively contribute to "the establishment of a specific chromatin structure"

  • The remodeling complexes SIRT7 interacts with control nucleosome positioning and chromatin accessibility

• Genomic targeting:

  • SIRT7 has been shown to occupy tRNA genes and interact with Pol III and TFIIIC2

  • This suggests coordinated regulation of chromatin at specific genomic loci

These interactions position SIRT7 as a multifaceted regulator that can influence gene expression both through direct effects on transcription machinery and through modification of chromatin structure, linking epigenetic regulation to cellular metabolic state.

How can researchers validate SIRT7 antibody specificity in knockout/knockdown models?

Validating SIRT7 antibody specificity using genetic models is critical for ensuring reliable experimental results. The search results indicate several publications have used knockdown/knockout approaches for SIRT7 antibody validation . Researchers should implement the following comprehensive validation strategy:

• Generate appropriate SIRT7-deficient models:

  • SIRT7 knockout cell lines using CRISPR-Cas9 technology

  • SIRT7 knockdown cells using siRNA or shRNA approaches

  • SIRT7 knockout mouse tissues (if available)

• Western blot validation:

  • Run parallel samples from wild-type and SIRT7 KO/KD cells/tissues

  • Use the SIRT7 antibody at manufacturer's recommended dilution (typically 1:1000-1:4000)

  • A specific antibody should show absence or significant reduction of the 45 kDa band in KO/KD samples

  • Include loading controls (e.g., β-actin, GAPDH) to ensure equal protein loading

• Immunohistochemistry validation:

  • Perform IHC on wild-type and SIRT7 KO/KD tissues using standard protocols

  • Compare staining patterns; specific antibodies should show absent or reduced nuclear/nucleolar staining in KO/KD samples

  • Include secondary antibody-only controls to assess background staining

• Positive controls:

  • Include cell lines known to express high levels of SIRT7 (e.g., HeLa, PC-3, HepG2)

  • These serve as positive controls to confirm antibody functionality

• Rescue experiments:

  • For additional validation, reintroduce SIRT7 expression in KO cells

  • Confirm restoration of antibody signal in rescued cells

This systematic validation approach ensures that observed signals genuinely represent SIRT7 protein rather than non-specific binding.

What are the recommended protocols for SIRT7 immunohistochemistry?

Based on the literature, the following optimized protocol is recommended for SIRT7 immunohistochemistry:

Sample preparation:

• Use paraffin-embedded tissue sections

• Mouse and rat liver tissues show reliable SIRT7 expression and can serve as positive controls

Detailed IHC protocol:

• Antigen retrieval:

  • Heat-mediated antigen retrieval is essential

  • Primary method: Use Citrate buffer (pH 6.0) for 20 minutes

  • Alternative method: TE buffer (pH 9.0) can also be effective

• Blocking and antibody incubation:

  • Apply primary antibody at recommended dilution:

    • For Abcam ab259968: 1/100 dilution

    • For Proteintech 12994-1-AP: 1:50-1:500 dilution range

  • Incubate for 30 minutes at room temperature

  • For detection, use a rabbit-specific IHC polymer detection kit with HRP/DAB (e.g., Abcam ab209101)

• Counterstaining:

  • Counterstain with Hematoxylin to visualize nuclei

• Controls:

  • Include secondary antibody-only controls (omit primary antibody)

  • Use known positive tissue samples (mouse/rat liver shows good nuclear staining)

• Expected results:

  • Nuclear staining pattern should be observed in positive cells

  • This pattern aligns with SIRT7's known localization and function

The search results indicate successful immunostaining has been performed using a Leica Biosystems BOND® RX instrument , though standard manual IHC procedures should also be effective.

What are the optimal dilutions for SIRT7 antibodies in different applications?

Based on extensive validation data, here are the recommended dilutions for SIRT7 antibodies across different applications:

Important optimization considerations:

• Sample-dependent factors:

  • The search results explicitly note that optimal dilution is "sample-dependent"

  • Manufacturers recommend that antibodies "should be titrated in each testing system to obtain optimal results"

• Cell/tissue type variations:

  • Human cell lines: HeLa, HEK-293T, HepG2, MCF7, PC-3

  • Mouse tissues/cells: Liver, kidney, spleen, Hepa1-6, NIH/3T3

  • Rat samples: Liver, C6 cells

• Detection system considerations:

  • For WB: Goat Anti-Rabbit IgG H&L (HRP) at 1/20000 dilution has been validated

  • For IP detection: VeriBlot for IP Detection Reagent (HRP) at 1/5000 dilution helps minimize detection of IP antibody bands

Titration experiments starting with the recommended ranges are advised for new experimental systems to determine the optimal antibody concentration that provides specific signal with minimal background.

How can researchers troubleshoot poor signal in SIRT7 western blots?

When encountering issues with SIRT7 detection in western blots, researchers should systematically address the following potential problems:

• Weak or absent signal:

  • Antibody concentration: Try a more concentrated antibody dilution (e.g., 1:1000 instead of 1:4000)

  • Sample preparation: Ensure complete lysis; SIRT7 is a nuclear protein, so nuclear extraction protocols may improve yield

  • Protein amount: Increase loading amount (20 μg is typically used in validated protocols)

  • Transfer conditions: Optimize transfer time/voltage for proteins in the 45 kDa range

  • Exposure time: Published protocols mention a 3-minute exposure time for SIRT7 detection

• High background:

  • Blocking optimization: Use 5% non-fat dry milk in TBST as validated in published protocols

  • Antibody dilution: Increase dilution if background is excessive

  • Washing steps: Extend wash times between antibody incubations

  • Secondary antibody: Ensure appropriate dilution (e.g., 1/20000 for HRP-conjugated anti-rabbit)

• Incorrect band size:

  • Expected size: SIRT7 should appear at 45 kDa (calculated: 44 kDa)

  • Positive controls: Include known positive samples (e.g., HeLa, HepG2 cells)

  • Protein degradation: Use fresh samples with protease inhibitors

  • Sample denaturation: Ensure complete denaturation before loading

• Validation controls:

  • Consider using SIRT7 knockdown/knockout samples as negative controls

  • Include positive control samples with confirmed SIRT7 expression:

    • Human: HeLa, HEK-293T, HepG2, MCF7, PC-3 cells

    • Mouse: Liver tissue, Hepa1-6, NIH/3T3 cells

    • Rat: Liver tissue, C6 cells

When troubleshooting, change only one variable at a time and document all modifications to identify the specific factor affecting SIRT7 detection.

What controls should be included when using SIRT7 antibodies?

To ensure experimental rigor when using SIRT7 antibodies, researchers should incorporate these essential controls:

• Positive controls:

  • Cell lines: PC-3, HeLa, HEK-293T, HepG2, or MCF7 cells for human studies

  • Mouse samples: Liver tissue, kidney tissue, Hepa1-6, or NIH/3T3 cells

  • Rat samples: Liver tissue or C6 cells

  • These samples have confirmed SIRT7 expression in published literature

• Negative controls:

  For Western Blot:

  • SIRT7 knockdown/knockout samples (if available)

  • The search results mention published studies using KD/KO validation

  For Immunohistochemistry:

  • Secondary antibody only control (omit primary antibody)

  • As described in the protocols: "Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB"

  For Immunoprecipitation:

  • IgG control antibody from the same species as the SIRT7 antibody

  • Protocol specifies using "1 μg of IgG control antibody" in parallel with "1 μg of SIRT7 antibody"

• Loading controls for Western Blot:

  • Housekeeping proteins (β-actin, GAPDH) to ensure equal loading

  • Nuclear markers (e.g., Lamin B) when analyzing nuclear fractions

• Application-specific controls:

  For co-immunoprecipitation studies:

  • Input controls (typically 5-10% of the lysate used for IP)

  • Protocol specifies: "Reserve a 50 μL aliquot in a separate new 1.5 mL tube for input controls"

Proper implementation of these controls ensures the reliability and reproducibility of experimental results, particularly important given SIRT7's roles in multiple cellular processes and emerging connections to disease states.

How should antigen retrieval be performed for optimal SIRT7 detection in IHC?

Antigen retrieval is critical for successful SIRT7 immunohistochemistry. Based on published protocols, the following specific methods are recommended:

Primary recommended method:

• Buffer: Citrate buffer (pH 6.0)

  • Also referred to as "epitope retrieval solution 1" in Leica Biosystems systems

• Duration: 20 minutes

• Temperature: Heat-mediated (typically 95-100°C)

• Validation: This method has been specifically validated for successful nuclear SIRT7 staining in mouse and rat tissues

Alternative method:

• Buffer: TE buffer (pH 9.0)

• Note: This is suggested as an alternative for the Proteintech antibody (12994-1-AP)

• Usage context: "Suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0"

Protocol implementation:

• Deparaffinize and rehydrate tissue sections

• Immerse slides in preheated retrieval buffer (Citrate pH 6.0 or TE pH 9.0)

• Heat at 95-100°C for 20 minutes

• Allow slides to cool in buffer for 20 minutes

• Wash in PBS before proceeding with IHC protocol

Expected outcomes:

• Proper antigen retrieval results in clear nuclear staining in positive cells

• When performed correctly, mouse and rat liver tissues show distinct nuclear SIRT7 staining patterns

• Published results cite PMID: 16618798 as reference for expected staining patterns

Complete and effective antigen retrieval is particularly important for nuclear proteins like SIRT7, as formaldehyde fixation can mask epitopes through extensive protein cross-linking.