SEL1L Antibody, Biotin conjugated

What is SEL1L and why is it important to study in molecular research?

SEL1L is an endoplasmic reticulum-resident transmembrane protein with a calculated molecular weight of 89 kDa that plays critical roles in multiple cellular processes. It functions primarily as an adaptor protein for the HRD1 ubiquitin ligase in the endoplasmic reticulum-associated degradation (ERAD) pathway, which is essential for protein quality control . The protein contains a Fibronectin-2 (FN2) domain on its luminal face that binds collagen, allowing SEL1L to function as a sensor of collagen biosynthesis . Research has demonstrated that SEL1L is indispensable for proper ERAD function, and its deletion leads to significant cellular stress, reduced translation, and formation of ribosomal aggregates . These characteristics make SEL1L a crucial target for studying protein quality control mechanisms, ER stress responses, and various pathologies associated with protein misfolding.

What experimental applications are recommended for biotin-conjugated SEL1L antibodies?

Based on data from multiple SEL1L antibodies, biotin-conjugated versions would be suitable for the following validated applications:

While the search results specifically mention unconjugated antibodies, biotin conjugation generally enhances detection sensitivity through streptavidin-based amplification systems while maintaining the same application versatility . When using biotin-conjugated antibodies, researchers should be aware that endogenous biotin in some tissues may cause background issues, necessitating appropriate blocking steps.

What species reactivity can I expect with SEL1L antibodies, and how does this affect experimental design?

SEL1L antibodies demonstrate cross-reactivity with multiple species as shown in the following data:

How can I optimize detection of SEL1L-HRD1 complexes using biotin-conjugated SEL1L antibodies?

Detection of SEL1L-HRD1 complexes requires careful consideration of experimental conditions to preserve native protein interactions. Research has shown that the SEL1L-HRD1 interaction is critical for forming a functional complex, and mutations like S658P can reduce this interaction by 5-fold . When using biotin-conjugated SEL1L antibodies for this purpose, implement the following methodological approaches:

• Use mild detergent conditions (0.5% Nonidet P-40) for cell/tissue lysis to preserve protein-protein interactions .

• Consider crosslinking approaches before lysis to stabilize transient interactions.

• For co-immunoprecipitation experiments, a sequential approach may be beneficial:

  • First immunoprecipitate with anti-HRD1 antibody

  • Then detect with biotin-conjugated SEL1L antibody and streptavidin-HRP

  • Compare results with reverse approach (IP with SEL1L, detect HRD1)

Importantly, research has demonstrated that mutations in SEL1L can affect protein stability and interactions. When studying variant forms of SEL1L, expression levels of both SEL1L and HRD1 should be carefully monitored as their stability is interdependent . The biotin conjugation provides enhanced sensitivity for detecting these potentially reduced interactions through signal amplification with streptavidin systems.

What controls are essential when studying SEL1L in ERAD pathways using biotin-conjugated antibodies?

When investigating SEL1L's role in ERAD pathways, several controls are methodologically essential:

• Positive controls: Include known ERAD substrates such as:

  • Alpha-1 antitrypsin NHK-GFP (documented in MEF models)

  • IRE1α (shown to accumulate in SEL1L-deficient cells)

  • OS9 and CD147 (stabilized in SEL1L mutant cells)

  • Pro-arginine vasopressin mutant (Gly57Ser)

• Negative controls:

  • SEL1L knockout or knockdown cells (essential for antibody specificity validation)

  • Non-ERAD pathway proteins to confirm specificity of observed effects

• Technical controls for biotin conjugation:

  • Streptavidin-only controls to assess endogenous biotin background

  • Biotin blocking steps in tissue samples with high endogenous biotin

  • Isotype-matched irrelevant biotin-conjugated antibody

Research has shown that SEL1L depletion leads to accumulation of ERAD substrates, reduced translation, and formation of ribosomal aggregates, providing multiple readouts to confirm phenotypes . When analyzing Western blot data, be aware that loss of SEL1L function can lead to high molecular weight aggregates that may remain in the stacking gel, requiring careful gel preparation and analysis protocols .

How can I use biotin-conjugated SEL1L antibodies to investigate stress granule formation and regulation?

Recent research has revealed that the HRD1-SEL1L ubiquitin ligase complex specifically regulates the homeostasis of heat shock-induced stress granules (SGs) through the ubiquitin-proteasome system . To leverage biotin-conjugated SEL1L antibodies for studying this phenomenon, implement the following methodological approach:

• Co-localization studies:

  • Use biotin-conjugated SEL1L antibody with streptavidin-fluorophore detection

  • Co-stain with established SG markers such as G3BP1 (Proteintech #13057-2-AP) and TDP43 (ABclonal #A1183)

  • Apply heat shock treatment (42°C for 1 hour is standard) to induce SG formation

• Functional analysis:

  • Compare SG formation in wild-type versus SEL1L-depleted cells

  • Quantify SG number, size, and SEL1L co-localization

  • Monitor ubiquitination status of SG components using K48-linkage and K63-linkage specific polyubiquitin antibodies

• Temporal dynamics:

  • Design time-course experiments to capture SEL1L involvement in SG assembly and disassembly phases

When analyzing results, consider that SEL1L functions as part of a ubiquitin ligase complex that may regulate the degradation of SG components. Therefore, changes in SG dynamics in SEL1L-depleted cells might reflect both direct regulation and indirect effects through accumulated ERAD substrates. The biotin-streptavidin detection system provides superior sensitivity for detecting potentially subtle localization changes under stress conditions.

What approaches can I use to study SEL1L mutations or variants with biotin-conjugated antibodies?

Research has shown that SEL1L variants, such as S658P, can significantly impact protein function while having variable effects on protein expression levels . When studying SEL1L mutations, biotin-conjugated antibodies offer enhanced detection sensitivity, which is particularly valuable for variants with reduced expression. Implement the following methodological strategies:

• Expression analysis:

  • Compare wild-type and variant SEL1L protein levels via Western blot

  • Use biotin-conjugated antibodies with streptavidin-HRP for enhanced sensitivity

  • Quantify relative expression levels against loading controls

• Protein stability assessment:

  • Perform cycloheximide chase experiments to determine half-life differences

  • Research shows SEL1L S658P variant has reduced stability (see data below)

  Protein	Wild-type half-life	S658P variant half-life	Reduction
  SEL1L	Standard	Reduced by ~20%	Moderate
  HRD1	Standard	Reduced by ~30%	Moderate

• Functional readouts:

  • Monitor accumulation of known ERAD substrates (IRE1α, OS9, CD147)

  • Assess formation of high molecular weight aggregates

  • Evaluate protein translation rates in variant-expressing cells

Research with the SEL1L S658P variant demonstrated partial embryonic lethality (~50%) in a mixed genetic background, highlighting the critical nature of proper SEL1L function . When designing experiments with SEL1L variants, consider both heterozygous and homozygous models to capture potential dosage effects, as SEL1L haploinsufficiency may produce subtle phenotypes requiring sensitive detection methods.

How can I troubleshoot non-specific binding when using biotin-conjugated SEL1L antibodies?

Non-specific binding is a common challenge with antibody-based detection, particularly with biotin-conjugated antibodies due to endogenous biotin. Implement these methodological approaches to minimize background:

• Sample preparation optimization:

  • Test different fixation methods for ICC/IHC (paraformaldehyde versus methanol)

  • Optimize antigen retrieval conditions - SEL1L antibodies show best results with TE buffer pH 9.0

  • Implement proper blocking of endogenous biotin using commercial avidin/biotin blocking kits

• Antibody validation strategies:

  • Perform peptide competition assays using the immunogen (amino acids 22-190 of human SEL1L for some antibodies)

  • Include SEL1L knockout or knockdown samples as negative controls

  • Test multiple antibody dilutions to find optimal signal-to-noise ratio

• Signal verification:

  • Confirm the observed molecular weight matches the expected 89 kDa for SEL1L

  • Be aware that high molecular weight forms may appear in the stacking gel in samples with SEL1L dysfunction

  • Verify subcellular localization is consistent with ER residence

For Western blot applications, researchers have reported optimal results using dilutions between 1:1000-1:8000, though this requires optimization for each experimental system . For immunofluorescence applications, 1:100 dilution has been validated in multiple cell lines including L929 and C6 . Always perform antibody titration experiments to determine the optimal concentration that maximizes specific signal while minimizing background.