ssr2 Antibody

What is SSR2 and why is it an important research target?

SSR2 (Signal Sequence Receptor Beta, also known as Translocon-Associated Protein Beta or TRAP-Beta) is a 22-kD glycoprotein that forms a crucial subunit of the signal sequence receptor (SSR) complex. This glycosylated endoplasmic reticulum (ER) membrane receptor plays a vital role in protein translocation across the ER membrane. The SSR complex consists of two primary subunits: a 34-kD glycoprotein (alpha-SSR or SSR1) and the 22-kD SSR2 protein. The human beta-signal sequence receptor gene maps to chromosome bands 1q21-q23 . Research interest in SSR2 has increased as its involvement in fundamental cellular processes becomes better understood, particularly in relation to protein processing pathways and potential implications in disease states.

How do SSR2 antibodies differ from other antibodies targeting ER proteins?

SSR2 antibodies are specifically designed to recognize epitopes on the Signal Sequence Receptor Beta protein, distinguishing it from other ER-associated proteins. Unlike antibodies targeting broader ER markers (such as calnexin or BiP/GRP78), SSR2 antibodies provide precise detection of this specific translocon component. This selectivity enables researchers to investigate the distinct role of SSR2 in the protein translocation machinery. Available SSR2 antibodies include those targeting specific regions such as N-terminal domains (amino acids 18-145), C-terminal regions (amino acids 154-183), or other specific epitopes within the protein sequence . This epitope diversity allows researchers to select antibodies appropriate for their specific experimental questions regarding SSR2 localization, interaction, or function.

What are the validated applications for SSR2 antibodies in cellular research?

SSR2 antibodies have been validated for multiple research applications that enable investigation of this protein's expression, localization, and function. Western blotting (WB) applications typically employ dilutions ranging from 1:500 to 1:1000 to detect the approximately 22kDa SSR2 protein in cell and tissue lysates . Immunohistochemistry on paraffin-embedded sections (IHC-P) requires antigen retrieval (typically using 10mM citrate buffer, pH 6.0) and antibody dilutions of 1:50 to 1:200 . Immunofluorescence/Immunocytochemistry (IF/ICC) applications utilize similar dilutions (1:50 to 1:200) to visualize SSR2's endoplasmic reticulum localization . Additionally, these antibodies have been validated for flow cytometry (FACS) and ELISA applications, providing researchers with a comprehensive toolkit for investigating SSR2 across multiple experimental platforms .

How should I optimize Western blot protocols for SSR2 detection?

For optimal Western blot detection of SSR2, a methodical approach addressing sample preparation, antibody selection, and detection parameters is essential. Begin with proper sample preparation: lyse cells in a buffer containing appropriate protease inhibitors, and load approximately 25μg of protein per lane . For primary antibody incubation, use anti-SSR2 antibody at a 1:1000 dilution (for polyclonal antibodies) or at manufacturer-recommended concentrations for monoclonal variants . Following primary antibody incubation, employ a compatible secondary antibody system, such as HRP-conjugated anti-rabbit IgG (at 1:10,000 dilution if using a rabbit primary antibody) . For blocking, 3% non-fat dry milk in TBST has been validated to reduce background while maintaining specific signal . If detecting multiple proteins simultaneously, note that SSR2 has a calculated molecular weight of approximately 20kDa but typically migrates at an observed 22kDa position in SDS-PAGE gels . This slight discrepancy is likely due to post-translational modifications and should be accounted for when interpreting results.

What tissue and cell types are most appropriate for studying SSR2 expression?

SSR2 expression can be effectively studied across multiple tissue and cell types, with certain models providing particularly robust expression for experimental analysis. Several cell lines have been validated for SSR2 research, including HepG2 (human liver cancer), A-549 (human lung adenocarcinoma), and SKOV3 (human ovarian cancer) cells . In tissues, liver and pancreas from both mouse and rat models show strong SSR2 expression and serve as positive controls for antibody validation . Brain tissue has also been successfully used for immunohistochemical detection of SSR2 . When selecting appropriate models, consider that SSR2's endoplasmic reticulum localization is conserved across species, making comparisons between human, mouse, rat, and other organisms (including zebrafish, cow, guinea pig, horse, bat, hamster, monkey, and pig) feasible with antibodies demonstrating cross-reactivity . This cross-species reactivity enables evolutionary and comparative studies of SSR2 function across diverse experimental models.

How can I distinguish between SSR2 and other members of the translocon complex in co-immunoprecipitation experiments?

Distinguishing between SSR2 and other translocon components in co-immunoprecipitation (co-IP) experiments requires careful antibody selection and experimental controls. First, select an SSR2 antibody with minimal cross-reactivity to other translocon components, particularly SSR1 (the alpha subunit), which shares functional similarities. Antibodies targeting unique regions such as the C-terminus of SSR2 (amino acids 154-183) are ideal as they minimize cross-reactivity. Implement reciprocal co-IP approaches, performing parallel experiments with antibodies against other translocon components and comparing interaction profiles. Include stringent washing steps to reduce non-specific binding, and validate results using Western blotting with antibodies targeting distinct epitopes from those used in the immunoprecipitation. Finally, consider using mass spectrometry as an antibody-independent method to confirm the identity of co-precipitated proteins, which can definitively distinguish between SSR2 (22kDa) and other translocon components of different molecular weights.

How should I interpret conflicting data regarding SSR2's role in DNA replication versus protein translocation?

The literature contains an apparent discrepancy regarding SSR2's primary cellular function, with some sources describing involvement in DNA replication and repair processes while others emphasize its role in protein translocation across the ER membrane . To resolve this conflict, first examine the experimental evidence supporting each claim. The protein translocation function is well-established and supported by multiple independent studies identifying SSR2 as part of the translocon-associated protein complex (TRAP) in the ER membrane. The proposed DNA replication/repair function appears less substantiated in the primary literature and may result from confusion with similarly named proteins or recent unpublished findings. To clarify SSR2's functions in your experimental system, consider conducting subcellular fractionation experiments followed by Western blotting to confirm SSR2's predominant localization in ER fractions rather than nuclear fractions. Perform co-localization experiments using immunofluorescence microscopy with established ER markers (calnexin, PDI) and DNA replication/repair markers (PCNA, γH2AX). Finally, employ functional assays specific to each proposed role, such as in vitro translocation assays and DNA replication/repair assays, to determine which functions SSR2 directly influences in your system.

What are the critical parameters for quantitative analysis of SSR2 expression in disease models?

Quantitative analysis of SSR2 expression in disease models requires careful attention to several methodological parameters to ensure reliable and reproducible results. First, standardize sample collection and processing protocols across all experimental groups to minimize technical variability. For protein-level quantification via Western blotting, normalize SSR2 signal to appropriate housekeeping proteins that remain stable in your disease model (verify multiple loading controls such as β-actin, GAPDH, and α-tubulin). When using immunohistochemistry for tissue analysis, employ digital image analysis with consistent acquisition parameters, and quantify staining intensity using appropriate software with region-of-interest standardization. For transcriptional analysis, design qPCR primers spanning exon-exon junctions to avoid genomic DNA amplification, and validate primer efficiency using standard curves. Regardless of the quantification method, include multiple biological and technical replicates, and perform power analyses to ensure adequate sample sizes for detecting biologically meaningful differences. Consider that SSR2, as an ER protein, may show altered expression patterns in diseases involving ER stress or secretory pathway dysfunction, and interpret results in this context, potentially with parallel analysis of ER stress markers (XBP1 splicing, BiP/GRP78 levels).

What are the optimal storage and handling conditions to maintain SSR2 antibody activity?

Maintaining SSR2 antibody activity requires precise storage and handling protocols tailored to antibody format and experimental timeline. For short-term storage (up to one month), store antibodies at 4°C in recommended buffer formulations, typically PBS with stabilizers such as glycerol (50%) and preservatives like sodium azide (0.02%) . For long-term storage, aliquot antibodies to minimize freeze-thaw cycles and store at -20°C . When preparing working dilutions, use freshly prepared buffers at appropriate pH (typically 7.2-7.4) and include stabilizing proteins (BSA or non-animal alternatives) if diluting below recommended concentrations. Critically, avoid repeated freeze-thaw cycles, as these can significantly reduce antibody activity through protein denaturation and aggregation . If decreased performance is observed after storage, verify antibody concentration using absorbance measurements and consider testing with validated positive control samples before troubleshooting experimental systems. For applications requiring conjugated antibodies, note that fluorophore-labeled variants may require protection from light during all handling steps to prevent photobleaching.

How can I address background issues in immunohistochemistry with SSR2 antibodies?

Addressing background issues in SSR2 immunohistochemistry requires systematic optimization of multiple protocol parameters. First, optimize blocking conditions by testing different blocking agents (3-5% BSA, normal serum matching the secondary antibody host, or commercial blocking reagents) and extending blocking time to 1-2 hours at room temperature. For SSR2 antibodies, 3% non-fat dry milk in TBST has proven effective in Western blotting applications and may be adapted for IHC . Thoroughly evaluate antibody dilutions, testing a broader range than the recommended 1:50-1:200 , as optimal dilutions can vary based on tissue fixation and processing methods. Enhance antigen retrieval effectiveness by comparing heat-induced epitope retrieval methods (microwave, pressure cooker, or water bath) using 10mM citrate buffer (pH 6.0) or alternative buffers (EDTA, pH 8.0). If non-specific nuclear staining occurs, add 0.3% Triton X-100 during primary antibody incubation to improve membrane permeability and antibody access to the ER-localized SSR2. For paraffin-embedded tissues, ensure complete deparaffinization and consider extending washing steps between protocol stages. Finally, if background persists, implement a biotin-avidin blocking step if using biotin-based detection systems, or switch to polymer-based detection methods which typically offer improved signal-to-noise ratios.

How should I validate the specificity of SSR2 antibodies in my experimental system?

Validating SSR2 antibody specificity requires implementing multiple complementary approaches to ensure reliable experimental results. Begin with positive and negative control samples: use tissues or cell lines with known SSR2 expression (hepatocytes, pancreatic cells) as positive controls, while employing appropriate negative controls (secondary antibody only, isotype controls). For definitive validation, implement genetic approaches using siRNA/shRNA knockdown or CRISPR-Cas9 knockout of SSR2, verifying reduced signal with your antibody. Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide/recombinant protein (specific sequences targeting amino acids 18-145 of human SSR2) , which should abolish specific signals. Compare results from multiple antibodies targeting different SSR2 epitopes (N-terminal vs. C-terminal) to confirm consistent localization and expression patterns. For Western blotting applications, verify that the detected protein matches the expected molecular weight (calculated 20kDa, observed 22kDa) and exhibits appropriate subcellular fractionation (enriched in ER membrane fractions). Finally, consider orthogonal detection methods such as RNA-seq or proteomics to correlate protein detection with transcript levels and mass spectrometry identification.

SSR2 Antibody Comparison Table

Protocol for Optimized Western Blot Detection of SSR2

• Sample Preparation:

  • Lyse cells in RIPA buffer with protease inhibitors

  • Quantify protein concentration using Bradford or BCA assay

  • Load 25μg protein per lane

• Electrophoresis:

  • Use 12-15% SDS-PAGE gels (optimal for 22kDa proteins)

  • Run at 100V through stacking gel, 150V through resolving gel

• Transfer:

  • Transfer to PVDF membrane at 100V for 1 hour or 30V overnight at 4°C

• Blocking:

  • Block with 3% non-fat dry milk in TBST for 1 hour at room temperature

• Primary Antibody:

  • Incubate with anti-SSR2 antibody at 1:1000 dilution in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Washing:

  • Wash 3x for 10 minutes each with TBST

• Secondary Antibody:

  • Incubate with HRP-conjugated secondary antibody (1:10,000)

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using ECL reagent

  • Expected band size: 22kDa (observed) vs. 20kDa (calculated)

SSR2 Protein Characteristics and Function Table

What are emerging applications of SSR2 antibodies in disease research?

SSR2 antibodies are increasingly being applied in disease research contexts that extend beyond basic protein characterization. As the role of ER stress and unfolded protein response (UPR) becomes better understood in conditions ranging from neurodegenerative disorders to cancer, SSR2 antibodies offer valuable tools for investigating translocon complex alterations in pathological states. In cancer research, examining SSR2 expression patterns across tumor types may reveal correlations with disease progression or treatment resistance, particularly in cancers with elevated secretory demands. For neurodegenerative diseases characterized by protein misfolding (Alzheimer's, Parkinson's), SSR2 antibodies enable investigation of potential translocon dysfunction. In metabolic disorders, particularly diabetes, where ER stress plays a pathogenic role in pancreatic β-cell dysfunction, SSR2 antibodies permit analysis of translocon integrity under metabolic stress conditions. These applications require careful method optimization, including dual-labeling approaches with disease-specific markers and quantitative analysis techniques that maintain sensitivity while allowing for high-throughput screening of clinical samples.

How can I design experiments to investigate SSR2's role in the unfolded protein response?

Designing experiments to investigate SSR2's role in the unfolded protein response (UPR) requires multi-faceted approaches that connect SSR2 function to established UPR pathways. Begin with induction experiments using classical UPR inducers (tunicamycin, thapsigargin, DTT) across time courses, monitoring SSR2 expression changes at both protein (Western blot) and transcript (qRT-PCR) levels. Complement these with co-immunoprecipitation studies to identify dynamic interactions between SSR2 and key UPR sensor proteins (IRE1α, PERK, ATF6) under basal and stressed conditions. Implement SSR2 knockdown/knockout models using siRNA or CRISPR-Cas9 technology, then assess the impact on UPR activation by measuring downstream effectors (XBP1 splicing, ATF4 and CHOP expression, BiP/GRP78 induction). Employ high-resolution imaging techniques (super-resolution microscopy, proximity ligation assays) to visualize SSR2 redistribution during ER stress. For functional analysis, perform protein translocation assays using reporter constructs in control versus SSR2-depleted cells under normal and ER stress conditions. Finally, extend findings to disease-relevant models, comparing SSR2 behavior in cells from normal donors versus patients with diseases characterized by chronic ER stress, establishing potential connections between SSR2 dysfunction and pathological UPR activation.