SESN1 Antibody, Biotin conjugated

What is SESN1 and why is it an important research target?

SESN1 (Sestrin 1) is a stress-responsive protein that belongs to the evolutionarily conserved sestrin family. It is primarily regulated by the p53 tumor suppressor protein and plays critical roles in cellular responses to DNA damage and oxidative stress . SESN1 mediates p53-dependent inhibition of cell growth by activating AMP-activated protein kinase (AMPK), which subsequently inhibits the mammalian target of rapamycin (mTORC1) protein . Additionally, SESN1 serves as an important antioxidant defense mechanism by regenerating overoxidized peroxiredoxins . Recent studies have identified SESN1 as a potential tumor suppressor in lung and other cancer types, functioning through STAT3 signaling pathway suppression . Its involvement in age-related processes, particularly in skeletal muscle, and in conditions like hypertension makes it a valuable target for various biomedical research areas .

What are the key applications for SESN1 antibody in research?

Based on the available literature, SESN1 antibodies are primarily used in the following applications:

• Western Blotting (WB): Used for protein detection and quantification in tissue lysates and cell lines with recommended dilutions typically between 1:500-1:2000 .

• Immunohistochemistry (IHC): For detecting SESN1 expression in tissue sections, usually at dilutions of 1:50-1:200 .

• Enzyme-Linked Immunosorbent Assay (ELISA): For measuring SESN1 levels in serum and cell culture medium .

• Immunofluorescence (IF): For visualizing subcellular localization of SESN1 .

Biotin-conjugated versions of these antibodies would allow for enhanced detection sensitivity in these applications due to the strong interaction between biotin and streptavidin/avidin detection systems.

What is the molecular weight of SESN1 protein and what band should I expect in Western blot?

SESN1 has a calculated molecular weight of approximately 64 kDa (551 amino acids), but the observed molecular weight in Western blotting typically ranges between 66-70 kDa . This slight discrepancy between calculated and observed molecular weights may be due to post-translational modifications. When running Western blots for SESN1, researchers should expect to see bands in the 66-68 kDa range in human samples, with similar molecular weights observed in mouse and rat samples .

What is the difference between using a biotin-conjugated SESN1 antibody versus an unconjugated antibody?

While the search results don't specifically address biotin-conjugated SESN1 antibodies, the general advantages of biotin conjugation include:

• Enhanced sensitivity: The biotin-streptavidin system offers one of the strongest non-covalent biological interactions, providing amplified signal detection.

• Versatility: Biotin-conjugated antibodies can be used with various detection systems (HRP, fluorescent, or gold-conjugated streptavidin).

• Reduced background: The biotin-streptavidin system often produces cleaner results with less non-specific binding.

• Multiplexing capability: Biotin-conjugated primary antibodies can be used alongside unconjugated antibodies from the same host species in co-localization studies.

The unconjugated antibodies described in the search results require secondary antibody detection systems and are typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

How does SESN1 function in the regulation of cell death and proliferation?

Recent research has revealed that SESN1 plays crucial roles in regulating cell death and proliferation through both mTORC1-dependent and independent mechanisms:

• mTORC1-independent regulation: Studies in lung adenocarcinoma A549 cells have shown that inactivation of SESN1 accelerates cell proliferation and confers resistance to cell death without affecting mTORC1 activity .

• STAT3 pathway suppression: SESN1 regulates cell proliferation and death by suppressing the STAT3 transcription factor. SESN1 deficiency has been demonstrated to stimulate STAT3 by downregulating the PTPRD phosphatase, which is responsible for STAT3 dephosphorylation .

• Genotoxic stress response: Knockout of SESN1 in cells dramatically increases colony formation after treatment with genotoxic agents like cisplatin and etoposide, indicating that SESN1 plays a role in cell death regulation following DNA damage .

• MCL1 regulation: SESN1 deficiency leads to increased expression of the anti-apoptotic protein MCL1, and silencing MCL1 restores sensitivity to cisplatin in SESN1-knockout cells .

These findings suggest that SESN1 may function as a tumor suppressor by regulating cell proliferation and death through multiple pathways, making it an important target for cancer research.

How is SESN1 expression altered in pathological conditions and what are the implications for biomarker research?

SESN1 expression is dynamically regulated in several pathological conditions:

• Cancer: SESN1 is upregulated in response to genotoxic stress in cancer cells, and its inactivation confers resistance to chemotherapeutic agents, suggesting its potential role as a predictive biomarker for treatment response .

• Hypertension: Circulating SESN1 levels are increased in patients with hypertension, with statistical analysis showing negative correlation (β = -0.415, 95% CI = -0.636 to -0.194, p < 0.001) in simple linear regression and (β = -0.218, 95% CI = -0.304 to -0.132, p = 0.011) in multiple linear regression analyses . This suggests SESN1 might serve as a biomarker for hypertension and related cardiovascular conditions.

• Skeletal muscle aging: SESN1 has been identified as a FOXO3 effector that counteracts human skeletal muscle aging, indicating its potential as a biomarker for age-related muscle degeneration .

• Osteoarthritis: Suppression of SESN1 has been observed in aging and osteoarthritic cartilage, indicating dysfunction of an important stress defense mechanism .

When designing biomarker studies using biotin-conjugated SESN1 antibodies, researchers should consider these context-specific expressions and validate antibody performance in their specific pathological models.

What are the considerations for using SESN1 antibodies in multiplex immunofluorescence studies?

While the search results don't specifically address multiplex studies with SESN1 antibodies, researchers should consider the following when designing multiplex immunofluorescence experiments with biotin-conjugated SESN1 antibodies:

• Antibody compatibility: Ensure that the biotin-conjugated SESN1 antibody is compatible with other primary antibodies in terms of host species and isotype to avoid cross-reactivity.

• Epitope accessibility: Since SESN1 interacts with multiple proteins including components of the mTORC1 pathway and STAT3 signaling pathways , epitope masking may occur in certain cellular contexts. Antigen retrieval methods should be optimized accordingly.

• Signal separation: When using a biotin-conjugated SESN1 antibody alongside other fluorescent markers, ensure appropriate spectral separation and use sequential detection if necessary to prevent signal overlap.

• Controls: Include single-stained controls and isotype controls to validate specificity, especially important when studying SESN1 in complex tissues or in disease states where protein expression and interactions may be altered.

• Quantification methods: Develop robust quantification strategies, particularly for co-localization studies involving SESN1 and its interacting partners in the STAT3 and mTORC1 pathways .

What are the recommended protocols for using biotin-conjugated SESN1 antibody in Western blotting?

Based on the protocols for unconjugated SESN1 antibodies, the following adaptations would be appropriate for biotin-conjugated versions in Western blotting:

• Sample preparation: Prepare protein extracts from tissues (human skeletal muscle, testis, liver) or cell lines (HEK-293, HeLa, K-562) as these have shown positive SESN1 detection .

• Protein loading: Load 20-40 μg of total protein per lane based on the expression level of SESN1 in your sample.

• Antibody dilution: While unconjugated antibodies are typically used at 1:500-1:1000 dilutions , biotin-conjugated antibodies may require optimization starting at 1:1000 and adjusting based on signal intensity.

• Detection system: Use streptavidin-HRP (typically at 1:10,000-1:20,000 dilution) instead of secondary antibodies for detection.

• Expected result: Look for bands at 66-68 kDa, which corresponds to the observed molecular weight of SESN1 .

• Controls: Include both positive controls (human skeletal muscle tissue, HEK-293 cells) and negative controls (SESN1 knockout cells if available) to validate specificity .

• Stripping and reprobing: If performing multiple protein detections on the same membrane, stripping conditions should be optimized to maintain the biotin conjugate if redetection of SESN1 is planned.

How can SESN1 antibodies be used in ELISA to quantify serum levels in clinical samples?

ELISA protocols using SESN1 antibodies have been successfully employed to measure SESN1 levels in human serum and cell culture medium . For biotin-conjugated SESN1 antibodies in ELISA, consider the following methodology:

• Sandwich ELISA format: Use a capture antibody (unconjugated) against one epitope of SESN1 and the biotin-conjugated SESN1 antibody as the detection antibody against a different epitope.

• Sample preparation: For human serum samples, dilution optimization is essential as SESN1 levels vary in different pathological conditions, particularly in hypertension where elevated levels have been reported .

• Detection: Use streptavidin-HRP for detection of the biotin-conjugated antibody with 3,3',5,5'-tetramethylbenzidine (TMB) as substrate.

• Quantification: Prepare a standard curve using recombinant SESN1 protein to quantify the absolute SESN1 concentration in samples.

• Normalization: For cell culture medium samples, normalize SESN1 concentration to the number of nuclei or total protein content as previously described .

• Data analysis: When analyzing SESN1 in clinical samples, consider statistical approaches like those used in hypertension studies, including Spearman's correlation analysis for associations with clinical parameters and multiple linear regression analysis for multivariate relationships .

What are the optimal conditions for immunohistochemistry using SESN1 antibodies?

Based on the search results, immunohistochemistry (IHC) applications with SESN1 antibodies have been reported . For biotin-conjugated SESN1 antibodies in IHC, consider these recommendations:

• Dilution: Start with a dilution range of 1:50 to 1:200 as recommended for unconjugated antibodies , and optimize based on signal-to-noise ratio.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) is likely appropriate, though specific conditions may need optimization for different tissue types.

• Blocking: Include a biotin blocking step to prevent non-specific binding to endogenous biotin, particularly important in tissues with high biotin content (liver, kidney).

• Detection: Use streptavidin-HRP or streptavidin conjugated to a fluorophore for detection.

• Controls: Include positive control tissues such as skeletal muscle or liver and negative controls (primary antibody omission and isotype controls).

• Double staining: When combining with other antibodies for co-localization studies, consider sequential detection protocols to prevent cross-reactivity.

• Counterstaining: Use DAPI for nuclei visualization in fluorescent detection or hematoxylin for brightfield applications.

What are common issues when using SESN1 antibodies and how can they be resolved?

While the search results don't specifically address troubleshooting with SESN1 antibodies, based on the general information provided and common issues with antibody-based techniques, the following problems and solutions can be anticipated:

• Weak or no signal:

  • Increase antibody concentration

  • Extend incubation time

  • Optimize antigen retrieval (for IHC/IF)

  • Increase protein loading (for WB)

  • Check sample preparation for protein degradation

• Multiple bands in Western blot:

  • SESN1 has multiple isoforms due to alternative splicing

  • Use positive controls to identify the correct band (66-68 kDa)

  • Increase antibody specificity by using more stringent washing conditions

  • Consider using SESN1 knockout cells as negative controls

• High background:

  • For biotin-conjugated antibodies, include avidin/biotin blocking steps

  • Increase blocking time or try different blocking reagents

  • Increase washing stringency and duration

  • Reduce antibody concentration

  • For IHC, consider quenching endogenous peroxidase activity

• Inconsistent results between experiments:

  • Standardize sample collection and processing

  • Use internal loading controls

  • Prepare larger batches of antibody dilutions

  • Control for experimental variables that may affect SESN1 expression (stress conditions, cell density)

How should researchers validate the specificity of SESN1 antibodies in their experimental systems?

Proper validation of SESN1 antibodies is critical for reliable research outcomes. Researchers should:

• Perform knockout/knockdown validation:

  • Use SESN1 knockout cells as negative controls

  • Compare with SESN1 knockdown using siRNA or shRNA

  • Look for elimination or significant reduction of signal at the expected molecular weight (66-68 kDa)

• Conduct peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide (if available)

  • Observe elimination of specific signal

• Compare multiple antibodies:

  • Use antibodies targeting different epitopes of SESN1

  • Confirm consistent detection patterns

• Verify cellular localization pattern:

  • Compare observed subcellular distribution with published literature

  • Check for expected changes in localization under stress conditions, as SESN1 is stress-responsive

• Correlate with mRNA expression:

  • Compare protein detection with SESN1 mRNA levels by qPCR

  • Particularly important when studying conditions where SESN1 is transcriptionally regulated by p53

• Check reactivity across species:

  • If working with animal models, verify cross-reactivity with the species of interest (human, mouse, rat reactivity has been confirmed for some antibodies)

What considerations should be made when interpreting SESN1 expression data in different experimental contexts?

When interpreting SESN1 expression data, researchers should consider:

• Stress conditions: SESN1 is upregulated in response to various stressors, including genotoxic stress, oxidative stress, and DNA damage . Experimental conditions that induce cellular stress may affect baseline SESN1 levels.

• p53 status: Since SESN1 is regulated by p53 , the p53 status of the experimental system (wild-type, mutant, or null) will significantly impact SESN1 expression patterns.

• Cell type-specific expression: SESN1 expression varies across different tissues and cell types, with notable expression in skeletal muscle tissue, liver tissue, testis tissue, and certain cell lines (HEK-293, HeLa, K-562) .

• Pathological context: SESN1 expression is altered in various disease states including cancer, hypertension, and age-related conditions . The pathological context should be considered when interpreting expression changes.

• Post-translational modifications: Possible modifications may affect antibody recognition and apparent molecular weight.

• Relationship with other signaling pathways: SESN1 interacts with multiple signaling pathways including mTORC1 and STAT3 , which may confound interpretation if these pathways are also altered in the experimental condition.

• Technical variables: Sample preparation methods, detection systems, and quantification approaches can all influence the apparent expression levels of SESN1.

What are the emerging applications and future directions for SESN1 research?

Based on the current literature, several promising directions for SESN1 research are emerging:

• Therapeutic targeting: SESN1 reactivation represents a potential strategy for cancer treatment, particularly in tumors with aberrant STAT3 activation . Developing methods to restore or enhance SESN1 function could offer new therapeutic approaches.

• Biomarker development: The altered expression of SESN1 in conditions like hypertension and age-related muscle degeneration suggests its potential as a diagnostic or prognostic biomarker. Standardized assays for SESN1 detection in clinical samples will be important for translational research.

• Aging research: The role of SESN1 as a counteracting factor in skeletal muscle aging and its involvement in osteoarthritic cartilage highlight its importance in understanding and potentially intervening in age-related processes.

• Drug resistance mechanisms: The finding that SESN1 deficiency confers resistance to genotoxic agents like cisplatin warrants further investigation into how SESN1 status might predict treatment response in cancer.

• Metabolic regulation: The connection between SESN1 and mTORC1 signaling suggests further exploration of its role in metabolic disorders and potential therapeutic applications.

• Advanced imaging techniques: Development of improved methods for visualizing SESN1 protein interactions and subcellular localization in living cells will enhance our understanding of its dynamic functions.