SZT2 Antibody

What is SZT2 and why is it important in research?

SZT2 is a large protein (3432 amino acids) that forms part of the KICSTOR complex, which regulates mTORC1 signaling under different nutritional conditions. Its significance lies in its role as a negative regulator of mTORC1 signaling and its involvement in multiple cellular processes. Mutations in the SZT2 gene cause developmental and epileptic encephalopathy (DEE), characterized by seizures, intellectual disability, and macrocephaly . From a structural perspective, SZT2 contains a peroxisomal targeting signal (PTS1) and a predicted superoxide dismutase motif, though no enzymatic activity has been demonstrated for the latter .

Research on SZT2 is particularly important for understanding mechanisms underlying epilepsy and neurodevelopmental disorders. SZT2 knockout cells exhibit increased mTORC1 signaling activation (which can be reversed by Rapamycin or Torin treatments) and higher levels of autophagic components, suggesting its potential role in both mTORC1 regulation and autophagy .

How should I select an appropriate SZT2 antibody for my research?

When selecting an SZT2 antibody for research purposes, consider:

• Epitope targeting: Choose antibodies targeting well-conserved regions of SZT2. The C-terminus has been shown to contribute to mTORC1 signaling regulation, while residues 1026-1132 are crucial for binding to GATOR1 .

• Validation methods: Prioritize antibodies validated in multiple techniques relevant to your experimental design (Western blot, immunoprecipitation, immunofluorescence).

• Species reactivity: Ensure the antibody recognizes SZT2 in your species of interest.

• Controls: Plan to use appropriate controls in your experiments, including SZT2 knockout cells as negative controls .

The validation of SZT2 antibodies is particularly challenging due to the protein's large size and often low abundance in cells. In some studies, researchers have created FLAG-tagged SZT2 knockin cell lines to validate antibody specificity and optimize detection protocols .

What are the optimal conditions for detecting SZT2 protein via Western blotting?

Due to SZT2's large molecular weight (approximately 380 kDa) and typically low expression levels, detection via Western blotting requires specific optimization:

• Protein extraction: Use gentle lysis buffers containing protease inhibitors to prevent degradation. A buffer containing 50 mM HEPES [pH = 7.4], 150 mM NaCl, 2.5 mM MgCl₂, 10% glycerol has been used successfully in previous studies .

• Gel electrophoresis: Utilize low percentage (5-7%) polyacrylamide gels or gradient gels to facilitate proper separation of high molecular weight proteins.

• Transfer conditions: Implement extended transfer times (overnight at low voltage) with larger pore membranes (PVDF rather than nitrocellulose) to ensure complete transfer of the large protein.

• Signal enhancement: Consider using signal enhancers or high-sensitivity detection reagents due to potentially low abundance.

• Controls: Include positive controls (cells overexpressing SZT2) and negative controls (SZT2 knockout cells) to confirm antibody specificity .

• Protein loading: Load adequate amounts of protein (50-100 μg per lane) to ensure detection of low-abundance SZT2.

How can I optimize immunofluorescence protocols for low-abundance proteins like SZT2?

Standard immunofluorescence protocols often fail to detect endogenous SZT2 due to its low abundance. Based on published methodologies, the following optimization strategies are recommended:

• Signal amplification systems: Implement tyramide signal amplification (TSA) methods. For example, researchers have successfully used the Tyramide SuperBoost Kit (Invitrogen, B40941) to amplify SZT2 detection signals .

• Protocol modifications: After fixation and permeabilization, include a step with 3% hydrogen peroxide solution (1 hour incubation) to quench endogenous HRP activity before proceeding with primary antibody incubation .

• Extended antibody incubation: Increase primary antibody incubation time to 2+ hours at room temperature or overnight at 4°C, using optimized dilution ratios (typically 1:200 for anti-FLAG M2 antibody) .

• Thorough washing: Implement extended washing steps (3 times, 10 minutes each with 2 ml PBS per wash) to reduce background .

• Secondary detection system: For FLAG-tagged SZT2, use poly-HRP-conjugated secondary antibodies followed by tyramide working solution (3-minute incubation) to significantly enhance signal detection .

• Confocal microscopy: Utilize confocal microscopy with Z-stack acquisition to improve signal-to-noise ratio.

What approaches can I use to study SZT2 interaction with GATOR complexes?

SZT2 forms a complex with GATOR1 and GATOR2 components, collectively termed SOG (SZT2-orchestrated GATOR). To study these interactions:

• Size-exclusion chromatography (SEC): This technique has successfully revealed that SZT2 is enriched in fractions with a peak molecular weight around 1.06 MDa, close to the predicted size of a trimeric SZT2-GATOR1-GATOR2 complex (~1.03 MDa) .

• Crosslinking-assisted immunoprecipitation: This approach has validated SZT2-dependent GATOR1-GATOR2 interactions using total cell lysate as input. Notably, SZT2-GATOR2 interaction was lost in the absence of NPRL3, while SZT2-GATOR1 interaction was substantially diminished without WDR59, WDR24, and MIOS, suggesting cooperative binding .

• Sucrose density gradient centrifugation: This method has demonstrated that co-sedimentation of GATOR1 and GATOR2 components at high sucrose density was diminished in SZT2-deficient cells .

• Genetic approaches: Creating knockout lines for individual components helps establish dependency relationships. For example, SZT2-GATOR2 interaction was lost in NPRL3-deficient cells .

When designing these experiments, consider:

• Using mild detergents to preserve protein-protein interactions

• Including appropriate controls (knockouts of individual components)

• Verifying results with multiple approaches to confirm interactions

How can I establish SZT2 knockout and knockin cell lines for antibody validation?

Creating genetically modified cell lines for SZT2 research requires careful planning due to the gene's large size. Based on published protocols:

For SZT2 Knockout Lines:

• Design guide RNAs targeting SZT2 and clone into appropriate vectors (e.g., LentiCrisprV2) .

• Transfect cells and apply puromycin selection for approximately 3 days .

• Perform limited dilution to isolate single cell clones (approximately one cell per well in 96-well plates) .

• Expand clones after 2 weeks and screen by immunoblotting for SZT2 depletion .

• Validate knockout by sequencing the targeted genomic loci.

For SZT2 Knockin Lines (e.g., FLAG-tagged):

• Design guide RNA targeting the first coding exon of SZT2 (e.g., sgSZT2ATG) and clone into appropriate vectors .

• Generate double-stranded DNA containing your tag sequence (e.g., FLAG) with a linker (e.g., GG), flanked by 40 base pairs of sequence surrounding the SZT2 start codon .

• Include silent mutations to prevent retargeting of the modified locus .

• Transfect cells with both the guide RNA vector and the repair template.

• Select with puromycin and perform limited dilution cloning .

• Screen clones by immunoblotting with an antibody against your tag .

• Confirm positive clones by sequencing the targeted genomic loci .

This approach allows you to create robust cellular models for validating antibody specificity and studying SZT2 function under various conditions.

What is the relationship between SZT2 mutations and mitochondrial dysfunction in disease contexts?

Recent research has established a critical link between SZT2 mutations and mitochondrial energy metabolism dysregulation:

• OXPHOS pathway impairment: Fibroblasts from patients with compound heterozygous SZT2 variants show significantly reduced oxygen consumption rate (OCR), a key indicator of mitochondrial oxidative phosphorylation function .

• Quantifiable bioenergetic defects: Specific measurements reveal:

  • 29% decrease in basal respiration rate

  • 35% decrease in ATP-linked respiration

  • 48% decrease in maximal respiration capacity

  • Reduction of spare respiratory capacity to 19% of normal levels

• Glycolytic dysfunction: SZT2 variants also impair glycolytic activities with reduced basal glycolysis and compensatory glycolysis, preventing effective interplay between the two main energy pathways (OXPHOS and glycolysis) .

• mTORC1 connection: These metabolic findings align with the established link between mTORC1 activation and glycolytic enzyme gene expression .

When using SZT2 antibodies to study these disease mechanisms, researchers should consider examining both wild-type and mutant proteins in the context of mitochondrial localization and function. Analyzing patient-derived cells alongside genetically modified models can provide particularly valuable insights into pathophysiological mechanisms.

What cellular localization pattern should I expect when using SZT2 antibodies?

SZT2 primarily exhibits a lysosomal localization pattern, consistent with its role in nutrient sensing through the mTORC1 pathway. When performing immunofluorescence:

• Co-localization markers: SZT2 has been shown to co-localize with lysosomal markers including Lamp1 and Lamp2 . Contrary to some predictions based on sequence analysis (which identified a peroxisomal targeting signal), SZT2 did not co-localize with the peroxisomal marker PMP70 in experimental studies .

• Expected pattern: Expect a punctate cytoplasmic distribution that overlaps significantly with lysosomal markers.

• Control staining: When validating a new SZT2 antibody, perform co-staining with established lysosomal markers (Lamp1/Lamp2) to confirm the expected localization pattern .

• Nutrient response: Consider examining SZT2 localization under both nutrient-rich and starvation conditions, as the protein's involvement in nutrient sensing may affect its distribution.

• Technical challenge: Due to the low abundance of SZT2 protein, signal amplification methods are typically required for successful visualization .

How can I optimize SZT2 protein purification for biochemical studies?

Purifying SZT2 for biochemical analysis presents challenges due to its size and complex interaction network. Based on successful approaches:

• Expression system: For recombinant SZT2, establish stable cell lines expressing tagged versions (e.g., FLAG-SZT2) using lentiviral vectors like pLJM1 .

• Lysis conditions: Use gentle lysis buffers (e.g., 50 mM HEPES [pH = 7.4], 150 mM NaCl, 2.5 mM MgCl₂, 10% glycerol) with protease inhibitors to preserve protein integrity and interactions .

• Affinity purification: For FLAG-tagged SZT2, use anti-FLAG M2 affinity gel equilibrated with lysis buffer containing 0.2% Triton X-100 .

• Washing protocol: Wash beads four times with wash buffer containing 2.5 mM ATP, followed by an additional wash without ATP .

• Elution method: Elute bound proteins with wash buffer containing 0.5 mg/ml FLAG peptide for 30 minutes with gentle rotation .

• Filtration: Filter eluate through a 0.22 μm filter to remove any aggregates .

This protocol has been successfully used to purify SZT2 complexes including associated GATOR components for downstream analysis.

What are the critical controls when using SZT2 antibodies in co-immunoprecipitation experiments?

When performing co-immunoprecipitation (co-IP) to study SZT2 interactions:

• Negative controls:

  • IgG control: Perform parallel immunoprecipitation with isotype-matched IgG

  • Knockout control: Include SZT2 knockout cells to identify non-specific interactions

  • Peptide competition: Pre-incubate antibody with excess immunizing peptide to confirm specificity

• Input controls:

  • Analyze 2-5% of pre-IP lysate to confirm protein expression

  • Verify equal loading across experimental conditions

• Reciprocal co-IP:

  • Confirm interactions by immunoprecipitating presumed binding partners and blotting for SZT2

  • This is particularly important for validating interactions with GATOR complex components

• Crosslinking consideration:

  • For transient or weak interactions, consider using crosslinking agents before lysis

  • Crosslinking-assisted immunoprecipitation has been successfully used to validate SZT2-dependent GATOR1-GATOR2 interactions

• Nutrient condition controls:

  • Compare interactions under different nutrient conditions (fed vs. starved)

  • This is relevant as SZT2 functions within nutrient-sensing pathways

How can SZT2 antibodies contribute to understanding epilepsy mechanisms?

SZT2 mutations cause developmental and epileptic encephalopathy (DEE), making antibody-based research critical for uncovering disease mechanisms:

• Patient tissue analysis: SZT2 antibodies can be used to examine protein expression and localization in patient-derived samples, potentially revealing molecular differences between wild-type and mutant proteins.

• Animal model validation: In mouse models, SZT2 mutations have been shown to confer higher susceptibility to seizures in a semi-dominant manner . Antibody-based studies can help validate these models by confirming altered protein expression or localization.

• Mechanistic studies: By combining SZT2 antibodies with markers of neuronal activity or synaptic function, researchers can investigate how SZT2 dysfunction contributes to hyperexcitability in neuronal networks.

• Drug response monitoring: When testing potential therapeutics (e.g., mTOR inhibitors like Rapamycin), SZT2 antibodies can help monitor treatment effects on protein expression and downstream signaling pathways .

• Circuit-level analysis: In tissue sections, SZT2 antibodies can help identify which neuronal populations or brain regions are most affected by alterations in SZT2 function.

Understanding these mechanisms could potentially lead to more targeted therapeutic approaches for patients with SZT2-related epilepsy.

What role does SZT2 play in autophagy regulation and how can antibodies help investigate this function?

Recent interactome analysis revealed SZT2's unexpected connections to autophagy:

• Autophagy component elevation: SZT2 knockout cells exhibit higher levels of autophagic components, independent of the physiological conditions tested .

• Autophagy receptor interactions: Interactome data detected an enriched pool of selective autophagy receptors/regulators associated with SZT2 .

• Methodological approaches: To investigate SZT2's role in autophagy:

  • Use SZT2 antibodies alongside markers of autophagic flux (LC3, p62/SQSTM1)

  • Examine autophagosome formation and maturation in cells with different SZT2 expression levels

  • Assess SZT2 co-localization with autophagosomal markers under various nutrient conditions

  • Compare autophagy dynamics in wild-type versus SZT2 mutant or knockout cells

• Potential research questions: Key questions that can be addressed using SZT2 antibodies include:

  • Does SZT2 directly interact with core autophagy machinery?

  • Is SZT2 itself degraded by autophagy?

  • How do SZT2 mutations found in patients affect autophagic flux?

  • Can autophagy modulation rescue phenotypes in SZT2-deficient cells?

These investigations could reveal new therapeutic targets at the intersection of mTORC1 signaling and autophagy regulation.

How should I address common challenges when using SZT2 antibodies in experimental procedures?

When working with SZT2 antibodies, researchers commonly encounter several technical challenges:

• Low signal intensity in Western blots:

  • Solution: Increase protein loading (50-100 μg per lane)

  • Use high-sensitivity chemiluminescent substrates

  • Optimize primary antibody concentration and incubation time (overnight at 4°C)

  • Consider membrane pore size selection to accommodate SZT2's large molecular weight

• High background in immunofluorescence:

  • Solution: Extend blocking time (2+ hours)

  • Use specialized blocking agents (e.g., ImageiT FX Signal Enhancer)

  • Implement more extensive washing protocols

  • Optimize antibody dilutions systematically

• Non-specific bands in immunoblotting:

  • Solution: Always include SZT2 knockout controls

  • Consider using different antibodies targeting distinct epitopes

  • Optimize detergent conditions in lysis buffers

  • Implement gradient gels for better resolution of high molecular weight proteins

• Failed immunoprecipitation:

  • Solution: Test multiple lysis buffer compositions

  • Consider mild crosslinking to stabilize complexes

  • Use larger volumes of lysate given SZT2's low abundance

  • Consider affinity-tagged SZT2 expression systems for method optimization

• Inconsistent results across experiments:

  • Solution: Standardize cell culture conditions, especially nutrient status

  • Monitor mTORC1 activity status as a functional readout

  • Implement robust loading controls

  • Document lot numbers of antibodies and other key reagents