SEC16B Antibody

What is SEC16B and what cellular functions does it serve?

SEC16B (also known as LZTR2, RGPR-p117) is a mammalian homolog of Saccharomyces cerevisiae Sec16 that plays a crucial role in organizing transitional endoplasmic reticulum (ER) sites and regulating protein export from the ER . This peripheral membrane protein localizes to the ER membrane and Golgi apparatus membrane, where it functions as part of the cellular transport machinery . SEC16B specifically regulates the transport of peroxisomal biogenesis factors PEX3 and PEX16 from the ER to peroxisomes, making it essential for peroxisome formation and function .

Recent research has also implicated SEC16B in metabolic functions, with evidence suggesting associations between SEC16B single nucleotide polymorphisms and various measures of obesity . Furthermore, new findings indicate that mutations in SEC16B can disrupt collagen trafficking, leading to collagen accumulation in the ER and triggering increased ER stress, enhanced autophagosome formation, and higher levels of apoptosis . This connection to collagen trafficking links SEC16B to bone disorders resembling osteogenesis imperfecta, characterized by vertebral fractures, leg bowing, and short stature .

What applications are SEC16B antibodies validated for?

Commercial SEC16B antibodies have been validated for several laboratory applications, with specific performance characteristics varying by manufacturer and antibody clone. Based on the available data, most SEC16B antibodies are validated for the following applications:

For optimal results, researchers should perform application-specific titration experiments to determine the ideal antibody concentration for their specific experimental system . Multiple search results indicate that antibody performance can vary significantly depending on the sample type, preparation method, and detection system employed .

What is the expected molecular weight for SEC16B detection?

Researchers should be aware that there is a notable discrepancy between the calculated and observed molecular weights for SEC16B protein. This is critical information for accurately interpreting western blot results:

The consistent observation of SEC16B at a lower molecular weight than calculated (60-70 kDa vs. 117 kDa) suggests potential post-translational modifications, alternative splicing, or proteolytic processing . When validating a new SEC16B antibody, researchers should consider running appropriate positive controls such as mouse pancreas tissue or HeLa cells transfected with GFP-SEC16B to confirm the specificity of their detection .

How should antibody validation be approached for SEC16B detection?

Validating SEC16B antibodies requires a systematic approach due to the challenges associated with detecting endogenous SEC16B in many cell types. Based on published methodologies, the following validation workflow is recommended:

First, researchers should test the antibody against positive control samples known to express SEC16B, such as mouse pancreas tissue or human pancreatic cancer tissue, which have been confirmed to express detectable levels of the protein . For overexpression controls, transfecting cells with GFP-tagged SEC16B provides a reliable positive control for antibody validation, as multiple studies have successfully used this approach to confirm antibody specificity .

Second, knockdown validation using siRNA against SEC16B should be performed to confirm the specificity of any bands detected at the expected molecular weight. This step is crucial as some antibodies may detect non-specific bands that do not disappear upon SEC16B depletion . The literature indicates that this has been a challenge with several SEC16B antibodies, where observed bands were unaffected by siRNA-mediated suppression of SEC16B .

Third, cross-validation using multiple antibodies targeting different epitopes of SEC16B can provide additional confidence in detection specificity. Previous research has utilized this approach with antibodies targeting different regions (anti-SEC16B 996-1010, 883-897, and 393-408) to comprehensively evaluate specificity . This multi-epitope approach helps overcome potential limitations of individual antibodies.

What are common challenges in detecting endogenous SEC16B?

Detecting endogenous SEC16B has proven particularly challenging in cellular studies, with several specific issues identified in the literature:

Researchers have reported that multiple antibodies targeting different epitopes of SEC16B failed to detect endogenous protein in various cell types, despite successfully detecting overexpressed GFP-SEC16B . This suggests either very low endogenous expression levels below detection thresholds or cell type-specific expression patterns that may not include common laboratory cell lines . The research indicates that of three tested antibodies (anti-SEC16B 996-1010, anti-SEC16B 883-897, and anti-SEC16B 393-408), all could detect overexpressed GFP-SEC16B by western blotting, but none reliably detected endogenous protein .

Another significant challenge is non-specific binding, with antibodies detecting multiple bands on western blots. For instance, anti-SEC16B (393-408) detected multiple protein bands with faint signals around 100 kDa, anti-SEC16B (883-897) revealed several bands including three around 100 kDa, and anti-SEC16B (996-1010) detected a single band at ~60 kDa that did not correspond to the expected molecular weight and was unaffected by siRNA depletion .

For immunofluorescence applications, additional challenges arise. Some antibodies that perform well in western blotting may fail in immunofluorescence applications. For example, anti-SEC16B (393-408) was unable to detect GFP-SEC16B by immunofluorescence despite success in western blotting . Other antibodies showed diffuse staining patterns or detected puncta that did not colocalize with known ERES markers like Sec31A, suggesting non-specific labeling .

Which cell types and tissues are recommended for SEC16B studies?

Based on empirical evidence from multiple studies, researchers investigating SEC16B should focus on specific tissues and cell types where the protein has been reliably detected:

For researchers establishing new SEC16B detection protocols, mouse pancreas tissue represents the most consistently validated positive control sample for western blotting applications . When working with cell lines, creating stable cell lines expressing GFP-SEC16B (such as the HeLa GFP-SEC16B and RPE-1 GFP-SEC16B cell lines described in the literature) provides reliable model systems to study SEC16B function .

Notably, several common cell types including NHDF and C13NJ cells have shown inconsistent results with SEC16B antibodies, with diffuse staining patterns and puncta that did not correspond to genuine ERES (ER exit sites) . This suggests that careful selection of appropriate experimental systems is crucial for meaningful SEC16B studies.

How can SEC16B's role in collagen trafficking be experimentally assessed?

Recent discoveries linking SEC16B mutations to altered collagen trafficking present important research opportunities . To investigate this function, researchers can implement the following experimental approach:

First, establish appropriate cellular models. Patient-derived fibroblasts with SEC16B mutations provide an ideal system for studying the effects on collagen trafficking, as these have demonstrated accumulation of type I procollagen in the ER . Alternatively, CRISPR-Cas9 gene editing can be used to introduce specific SEC16B mutations in relevant cell types that express both SEC16B and collagen.

Second, implement multi-parameter assessment of collagen trafficking. This should include immunofluorescence microscopy to visualize collagen localization relative to ER and Golgi markers, western blotting to assess procollagen processing, and pulse-chase experiments to quantify trafficking kinetics. The research indicates that SEC16B mutations result in measurable collagen accumulation in the ER, providing a clear phenotypic readout .

Third, evaluate downstream consequences of trafficking defects. SEC16B-dependent collagen trafficking disruptions lead to several measurable cellular changes including increased ER stress, enhanced autophagosome formation, and elevated apoptosis levels . Quantifying these parameters using appropriate assays (e.g., ER stress markers, LC3 conversion for autophagy, and caspase activation for apoptosis) provides a comprehensive view of SEC16B's role.

For rescue experiments to confirm specificity, transfection of wild-type SEC16B into affected cells has been demonstrated to rescue the collagen trafficking defect, providing a powerful validation approach . The comprehensive experimental design should incorporate both morphological and biochemical readouts to fully characterize SEC16B's role in this process.

What strategies resolve conflicting results with different SEC16B antibodies?

When faced with discrepancies in results obtained using different SEC16B antibodies, researchers should implement a systematic troubleshooting approach:

First, conduct epitope mapping analysis. Different SEC16B antibodies target distinct epitopes (e.g., regions 996-1010, 883-897, and 393-408), which may be differentially accessible depending on protein conformation, interaction partners, or post-translational modifications . Comparing the specific epitopes recognized by each antibody can help interpret conflicting results.

Second, implement complementary detection methods. When antibody-based approaches yield inconsistent results, alternatives such as RNA-level detection (RT-qPCR), epitope tagging of endogenous SEC16B using CRISPR-Cas9 knock-in approaches, or mass spectrometry-based protein identification can provide orthogonal validation. The literature indicates that even when antibodies fail to detect endogenous SEC16B, GFP-tagged versions can be successfully visualized and studied .

Third, consider context-dependent protein expression and modification. SEC16B detection may be influenced by cell type-specific expression patterns, subcellular localization differences, or condition-dependent modifications. For example, research has shown that SEC16B localizes to puncta distributed throughout the cell in an ERES-like pattern when GFP-tagged and expressed in HeLa or RPE-1 cells . This characteristic localization pattern can serve as an additional validation criterion when evaluating antibody performance.

Fourth, utilize genetic approaches for validation. siRNA-mediated knockdown of SEC16B should reduce or eliminate specific bands in western blots if they genuinely represent SEC16B . The literature indicates that this approach has revealed non-specific detection in some cases, where presumed SEC16B bands were unaffected by siRNA depletion .

How can researchers investigate SEC16B's function in peroxisome biogenesis?

SEC16B regulates the transport of peroxisomal biogenesis factors PEX3 and PEX16 from the ER to peroxisomes, positioning it as a key player in peroxisome formation . To investigate this specialized function, researchers can implement the following experimental strategy:

First, establish appropriate visualization systems. Fluorescently tagged peroxisomal markers (such as GFP-SKL or RFP-PEX3) can be used alongside SEC16B antibodies or fluorescently tagged SEC16B to monitor spatial and temporal relationships during peroxisome biogenesis. This approach allows for live-cell imaging to track the dynamic process of peroxisome formation.

Second, implement trafficking assays to quantify PEX3 and PEX16 movement. Pulse-chase experiments with tagged PEX proteins can measure the rate of transport from the ER to peroxisomes. This can be complemented with photoactivatable or photoconvertible fluorescent proteins to specifically track newly synthesized populations of peroxisomal proteins.

Third, apply SEC16B manipulation approaches. These should include both loss-of-function (siRNA knockdown, CRISPR knockout) and gain-of-function (overexpression of wild-type or mutant SEC16B) strategies to determine how perturbing SEC16B levels affects PEX3 and PEX16 trafficking. The direct demonstration of SEC16B's role in regulating the transport of these specific factors provides a focused experimental readout .

Fourth, assess downstream consequences on peroxisome formation and function. This includes quantifying peroxisome number, size, and distribution through imaging approaches, as well as measuring peroxisomal enzyme activities (such as catalase or fatty acid β-oxidation) to determine functional consequences of SEC16B manipulation.

How should western blot protocols be optimized for SEC16B detection?

Optimizing western blot protocols for SEC16B detection requires attention to several critical parameters, based on empirical evidence from multiple studies:

Sample preparation is crucial for preserving SEC16B integrity. The recommended approach involves lysing cells or tissues in a buffer containing protease inhibitors, followed by immediate processing or flash freezing to prevent degradation . For tissue samples, mouse pancreas tissue has been validated as a reliable positive control that consistently expresses detectable SEC16B .

Regarding electrophoresis and transfer conditions, resolving SEC16B can be challenging due to its high molecular weight (calculated 71-117 kDa, observed 60-70 kDa) . Using gradient gels (4-12% or 4-15%) improves separation of high molecular weight proteins, while extended transfer times (overnight at low voltage or 2 hours at higher voltage) enhance transfer efficiency for large proteins.

Detection systems should be optimized based on expression levels. For weakly expressed endogenous SEC16B, enhanced chemiluminescence (ECL) substrates with higher sensitivity or fluorescence-based detection systems with lower background may be necessary. The literature indicates that even with optimized conditions, detecting endogenous SEC16B remains challenging in many cell types, suggesting very low natural expression levels .

What immunohistochemistry conditions are optimal for SEC16B detection?

Successful immunohistochemical detection of SEC16B requires careful optimization of several critical parameters:

Antigen retrieval methods significantly impact SEC16B detection in tissue samples. The recommended approach uses TE buffer at pH 9.0, which has been validated for detection in human pancreatic cancer tissue . As an alternative when this method proves insufficient, citrate buffer at pH 6.0 can be used . The selection between these methods should be empirically determined for each tissue type and fixation condition.

Antibody dilution ranges for IHC applications typically fall between 1:50 and 1:500, requiring experimental optimization for each tissue type and detection system . Generally, starting with a mid-range dilution (1:200) and adjusting based on signal-to-noise ratio is recommended. The literature emphasizes the importance of titrating the antibody in each testing system to obtain optimal results .

Blocking and incubation conditions should be optimized to minimize background staining while preserving specific signal. A standard approach involves blocking with 5-10% normal serum from the same species as the secondary antibody, followed by overnight primary antibody incubation at 4°C to maximize specific binding while minimizing non-specific interactions.

Detection systems should be selected based on the expected expression level and localization pattern. For SEC16B, which localizes to the ER membrane and Golgi apparatus membrane as a peripheral membrane protein, detection systems that provide subcellular resolution (such as tyramide signal amplification) may be beneficial for visualizing the characteristic punctate ERES-like distribution pattern observed in studies with tagged SEC16B .

How can researchers distinguish between SEC16B and the related SEC16A protein?

Differentiating between SEC16B and SEC16A is essential for specific functional studies, as these related proteins may have both overlapping and distinct roles in ER-to-Golgi transport. Researchers can implement several strategies to ensure specificity:

Antibody selection represents the primary approach for specific detection. Antibodies should be raised against unique sequences of SEC16B that do not share homology with SEC16A . The literature describes the development of three different anti-peptide antibodies (anti-SEC16B 996–1010, anti-SEC16B 883–897, and anti-SEC16B 393–408) designed against unique sequences to prevent cross-reaction with SEC16A .

Molecular weight discrimination provides an additional layer of specificity. SEC16B has a calculated molecular weight of 71-117 kDa and is typically observed at 60-70 kDa on western blots , while SEC16A is significantly larger. This size difference allows for discrimination when using properly calibrated western blots with appropriate molecular weight markers.

For functional studies, selective knockdown or knockout approaches can isolate SEC16B-specific effects. siRNA duplexes specifically targeting SEC16B have been validated in multiple studies . These can be used alongside SEC16A-specific siRNAs to determine the relative contributions of each protein to cellular processes of interest. The literature describes experiments where HeLa GFP-SEC16B cells were transfected with siRNA duplexes targeting SEC16A, allowing for the specific study of SEC16B functions in the absence of SEC16A .

Double-labeling immunofluorescence approaches can also help distinguish the proteins. Co-staining with antibodies against SEC16B and SEC16A, or using differentially tagged versions of these proteins, can reveal distinct localization patterns or differential responses to experimental manipulations, providing functional separation of these related proteins.

How can SEC16B antibodies be applied to obesity research?

SEC16B has been implicated in obesity through associations between its single nucleotide polymorphisms and various measures of obesity . This connection opens important avenues for research that can be facilitated by appropriate use of SEC16B antibodies:

For genetic association studies, SEC16B antibodies can help validate the functional consequences of identified polymorphisms. Researchers should design experiments comparing SEC16B protein expression, localization, and function in cells harboring different SEC16B genetic variants. This approach can help bridge the gap between genetic associations and molecular mechanisms.

In tissue-specific expression analysis, immunohistochemistry using validated SEC16B antibodies can identify differential expression patterns in metabolically relevant tissues (adipose, liver, hypothalamus) between lean and obese subjects . The recommended dilution range for IHC applications (1:50-1:500) should be optimized for each tissue type, with special attention to antigen retrieval conditions to ensure specific detection .

For mechanistic studies linking SEC16B to metabolic pathways, co-immunoprecipitation using SEC16B antibodies can identify novel protein-protein interactions that might explain its role in metabolism. This approach can reveal how SEC16B integrates with known metabolic signaling networks and potentially identify new therapeutic targets.

Cell culture models with manipulated SEC16B levels (overexpression or knockdown) can be used to study effects on insulin signaling, lipid metabolism, or adipocyte differentiation. SEC16B antibodies provide crucial tools for validating these manipulations and monitoring resulting changes in protein expression and localization.

What is the significance of SEC16B in collagen-related disorders?

Recent research has identified a critical role for SEC16B in collagen trafficking, with mutations leading to collagen accumulation in the ER and subsequent pathological changes reminiscent of osteogenesis imperfecta . This discovery opens important research avenues:

For clinical sample analysis, SEC16B antibodies can be used to assess SEC16B expression and localization in patient-derived cells or tissues from individuals with collagen-related disorders. Immunohistochemistry or immunofluorescence approaches using optimized antibody dilutions (1:50-1:500 for IHC) can reveal altered SEC16B distribution patterns that might contribute to disease pathology .

In mechanistic studies, co-localization analysis using SEC16B antibodies together with markers for collagen, ER stress, and autophagy can elucidate the sequence of cellular events that lead from SEC16B dysfunction to collagen accumulation and eventually to clinical manifestations . This multi-parameter imaging approach provides spatial and temporal insights into disease progression.

For therapeutic development targeting SEC16B-related disorders, SEC16B antibodies provide essential tools for screening and validating potential interventions. Compounds that restore normal collagen trafficking in SEC16B-mutant cells can be identified by monitoring changes in SEC16B and collagen localization patterns using appropriately validated antibodies.

The established connection between SEC16B mutations and features resembling osteogenesis imperfecta (including vertebral fractures, leg bowing, and short stature) highlights the potential clinical significance of SEC16B as a diagnostic marker and therapeutic target . SEC16B antibodies can enable both the fundamental research needed to understand these mechanisms and the translational studies required to develop interventions.