SSR2 Antibody

What is SSR2 and why is it significant in cellular research?

SSR2 (Signal Sequence Receptor, beta) is a 22-kD glycoprotein subunit of the signal sequence receptor complex involved in protein translocation across the endoplasmic reticulum (ER) membrane. It functions alongside the 34-kD glycoprotein alpha-SSR (SSR1) as part of the translocon-associated protein complex . The human SSR2 gene maps to chromosome bands 1q21-q23 and is implicated in viral mRNA translation and generic transcription pathways . Its significance lies in understanding protein trafficking mechanisms, ER stress responses, and its associations with conditions like calcaneonavicular coalition and osteosarcoma . Researchers targeting SSR2 can gain insights into fundamental cellular processes related to protein secretion and membrane protein biogenesis.

What are the key differences between polyclonal and monoclonal SSR2 antibodies?

Comparison of SSR2 Antibody Types:

Polyclonal antibodies, like the rabbit polyclonal described in source , recognize multiple epitopes on the SSR2 protein, enhancing detection sensitivity but potentially increasing background. Monoclonal antibodies, like clone PAT31G6AT or 31G6 , offer higher specificity by targeting a single epitope, improving experimental consistency but potentially limiting detection if that epitope is altered or masked .

How do I select the appropriate SSR2 antibody for my experimental design?

Selection of an appropriate SSR2 antibody requires consideration of several experimental factors:

• Target species reactivity: Different antibodies show varying reactivity profiles. Some antibodies like ABIN7270299 demonstrate cross-reactivity with human, mouse, and rat SSR2 , while others are human-specific .

• Experimental application: For Western blot applications, both polyclonal (SAB1401374) and monoclonal options are available, with recommended dilutions around 1μg/mL . For immunofluorescence studies, conjugated antibodies like NBP2-42647DL594 (DyLight 594-conjugated) offer direct visualization .

• Epitope accessibility: Consider the protein region recognized by the antibody. Some target the N-terminus, others the C-terminus, and some recognize internal sequences like amino acids 18-149 or 51-150 .

• Validation data: Review the validation data provided by manufacturers. For instance, source shows Western blot validation in human placenta and HeLa cells, while source provides immunocytochemistry validation data.

• Sample preparation method: If working with denatured samples (SDS-PAGE), ensure the antibody recognizes linear epitopes. For native conformations (immunoprecipitation), select antibodies validated for recognizing folded proteins.

What are the optimal conditions for using SSR2 antibodies in Western blot experiments?

For optimal Western blot detection of SSR2, researchers should consider the following protocol modifications:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors for efficient extraction

  • Heat samples at 70°C rather than 95°C to prevent SSR2 aggregation as it's a membrane protein

• Gel percentage:

  • Use 12-15% SDS-PAGE gels to resolve the ~22 kDa SSR2 protein effectively

• Transfer conditions:

  • Transfer at lower voltage (15V) overnight at 4°C for membrane proteins

  • Use PVDF membranes instead of nitrocellulose for better protein retention

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • For primary antibodies, dilutions vary by product:

    • SAB1401374: Use at 1 μg/mL concentration

    • H00006746-B01P: Experimentally determine optimal dilution, starting at 1:500

  • Incubate primary antibody overnight at 4°C for improved signal-to-noise ratio

• Detection controls:

  • Include human placenta or HeLa cell lysates as positive controls

  • Expected molecular weight: 22 kDa (may vary with post-translational modifications)

Multiple SSR2 antibodies have been validated for Western blot applications, with data showing successful detection in human placenta and HeLa cell extracts .

How can I optimize immunofluorescence protocols for SSR2 localization studies?

Achieving optimal immunofluorescence results for SSR2 localization requires:

• Fixation optimization:

  • 4% paraformaldehyde (15 minutes at room temperature) preserves ER structure

  • Avoid methanol fixation which can disrupt membrane protein epitopes

  • Include 0.1% saponin or 0.2% Triton X-100 for controlled permeabilization

• Antibody selection and dilution:

  • For direct detection: NBP2-42647DL594 (DyLight 594-conjugated monoclonal)

  • For indirect detection: Unconjugated antibodies (ABIN7270299 or unlabeled monoclonal)

  • Starting dilution of 1:500, then optimize based on signal strength

• Co-localization markers:

  • Include established ER markers (calnexin, PDI, or KDEL-tagged proteins)

  • For high-precision localization, super-resolution techniques like STORM or STED with appropriate fluorophore selection are recommended

• Signal enhancement:

  • Tyramide signal amplification for weak signals

  • Use mounting media with anti-fade agents to prevent photobleaching

• Validation approaches:

  • Perform Z-stack imaging to confirm ER membrane localization

  • Include siRNA knockdown controls to verify antibody specificity

The monoclonal antibody clone 31G6 has been specifically validated for immunofluorescence applications, showing the characteristic reticular ER pattern expected for SSR2 .

What are the critical considerations for using SSR2 antibodies in flow cytometry?

Flow cytometry analysis of SSR2 requires special considerations as it's primarily an ER membrane protein:

• Cell preparation protocols:

  • Gentle fixation with 2% paraformaldehyde (10 minutes)

  • Permeabilization is essential (0.1% saponin recommended for ER proteins)

  • Maintain cells at 4°C during processing to minimize internalization

• Antibody selection and staining:

  • Antibodies validated specifically for flow cytometry:

    • H00006746-B01P has demonstrated efficacy in flow cytometry

    • Clone 31G6 has also been validated for FACS applications

  • Use titrated antibody concentrations to determine optimal signal-to-noise

• Gating strategy:

  • Include viability dye to exclude dead cells

  • Use side scatter properties to eliminate debris

  • Compare with isotype controls matched to the specific antibody clone

• Validation and controls:

  • Positive controls: HEK293 cells overexpressing SSR2 show strong signal

  • Negative controls: Untransfected cells or SSR2 knockdown samples

  • Fluorescence minus one (FMO) controls are essential for accurate gating

• Data interpretation:

  • Expect heterogeneous expression levels reflecting varying ER content

  • Shifts in mean fluorescence intensity can indicate ER stress or altered protein trafficking

Flow cytometry analysis validated with antibody H00006746-B01P has successfully distinguished between negative control 293 cells and SSR2-expressing 293 cells , demonstrating the feasibility of this approach despite SSR2's intracellular localization.

How should researchers troubleshoot non-specific binding or high background when using SSR2 antibodies?

When encountering high background or non-specific binding with SSR2 antibodies, implement these systematic troubleshooting steps:

• Antibody-specific optimizations:

  • Titrate antibody concentration (start with a dilution series from 1:250 to 1:2000)

  • For polyclonal antibodies like ABIN7270299, pre-adsorption against cell lysates from non-relevant species may reduce cross-reactivity

  • Switch to more specific monoclonal options like clone 31G6 if polyclonal antibodies show high background

• Protocol modifications for Western blotting:

  • Increase washing duration and frequency (5x5 minutes with 0.1% Tween-20 in PBS)

  • Add 0.1-0.5% SDS to wash buffer for stringent conditions

  • Increase blocking agent concentration to 5-10% or switch between milk and BSA

  • For membrane proteins like SSR2, add 0.05% SDS to antibody dilution buffer

• Immunofluorescence/IHC optimizations:

  • Include 10% serum from the secondary antibody host species in blocking buffer

  • Extend blocking time to 2 hours at room temperature

  • Use commercially available background reducers specific to the detection system

  • For tissue sections, treat with 3% hydrogen peroxide to block endogenous peroxidase

• Technical validation approaches:

  • Run antibody validation with SSR2 knockdown samples

  • Include absorption controls using the immunizing peptide (amino acids 18-149 for some antibodies)

  • Compare signal patterns across multiple SSR2 antibodies recognizing different epitopes

• Flow cytometry-specific remedies:

  • Implement more stringent gating strategies

  • Include dead cell discrimination dyes

  • Reduce antibody concentration and extend incubation time

The quality of different SSR2 antibodies varies significantly. Protein-A purified antibodies like the one described in source typically offer improved specificity compared to crude serum preparations.

What controls are essential for validating SSR2 antibody specificity in different experimental contexts?

Essential controls for SSR2 antibody validation across experimental platforms:

For rigorous validation in research contexts:

• Genetic validation: Compare antibody signal between wild-type and CRISPR/Cas9 SSR2 knockout cell lines

• Orthogonal validation: Correlate protein detection with mRNA levels via RT-qPCR

• Cross-platform validation: Confirm consistent results across multiple techniques (e.g., if Western blot shows a single band at 22 kDa, immunofluorescence should show specific ER pattern)

• Species validation: When using antibodies across species, always validate with species-specific positive controls

When using monoclonal antibodies like clone 31G6 , epitope mapping becomes particularly important as these antibodies recognize a single determinant that may be masked in certain experimental conditions.

How do different fixation and sample preparation methods affect SSR2 antibody performance?

SSR2, as an ER membrane protein, is particularly sensitive to fixation and preparation methods:

• Chemical fixation comparison:

  • Paraformaldehyde (4%, 10-15 min): Preserves protein structure but may mask some epitopes

  • Methanol (-20°C, 10 min): Better for some epitopes but disrupts membrane structure

  • Acetone (-20°C, 5 min): Good for cytoskeletal preservation but can extract membrane proteins

  • Glutaraldehyde (0.1-0.5%): Excellent ultrastructure preservation but significant autofluorescence

  Recommendation: Start with 4% paraformaldehyde for most SSR2 applications, as it maintains ER morphology while preserving most epitopes recognized by validated antibodies .

• Antigen retrieval methods for tissue sections:

  • Heat-induced epitope retrieval (HIER): 10mM sodium citrate, pH 6.0, 95°C for 20 minutes

  • Enzymatic retrieval: Proteinase K treatment (20μg/mL, 15 minutes at room temperature)

  Recommendation: For formalin-fixed paraffin-embedded tissues, HIER with sodium citrate buffer generally yields optimal results for SSR2 detection.

• Permeabilization optimization for intact cells:

  • Triton X-100 (0.1-0.5%): Strong permeabilization, may extract some membrane proteins

  • Saponin (0.1-0.2%): Milder, reversible permeabilization, better for membrane proteins

  • Digitonin (10-50μg/mL): Selective permeabilization of plasma membrane

  Recommendation: Use 0.1% saponin for optimal preservation of SSR2 epitopes while allowing antibody access to the ER lumen.

• Sample storage considerations:

  • Fixed cells/tissues should not be stored for prolonged periods before antibody incubation

  • For paraffin blocks, freshly cut sections yield better results

  • Freeze-thaw cycles significantly impact membrane protein epitopes

When working with the monoclonal antibody clone PAT31G6AT (derived from immunization with recombinant human SSR2 protein 18-149 amino acids) , mild fixation and permeabilization conditions preserve optimal epitope recognition.

How can SSR2 antibodies be applied to study ER stress and the unfolded protein response?

SSR2 antibodies provide valuable tools for investigating ER stress and unfolded protein response (UPR) mechanisms:

• Quantitative changes in SSR2 expression:

  • Western blot analysis using antibodies like SAB1401374 or H00006746-B01P can quantify SSR2 upregulation during ER stress

  • Standardized protocol: Treat cells with tunicamycin (2μg/mL, 4-24 hours) or thapsigargin (500nM, 4-16 hours), then analyze SSR2 protein levels

  • Compare with established UPR markers (BiP/GRP78, CHOP, XBP1 splicing)

• Subcellular redistribution during stress:

  • Immunofluorescence with NBP2-42647DL594 enables visualization of SSR2 relocalization

  • Co-staining with UPR sensors (IRE1α, PERK, ATF6) reveals functional relationships

  • Quantitative analysis parameters: ER expansion, fragmentation, and perinuclear clustering

• Protein-protein interaction studies:

  • Immunoprecipitation with anti-SSR2 antibodies followed by mass spectrometry identifies stress-induced binding partners

  • Proximity ligation assays using antibody pairs targeting SSR2 and other translocon components

  • FRET/FLIM microscopy using labeled secondary antibodies to assess nanoscale interactions

• Functional assays:

  • Correlate SSR2 expression/localization with protein translocation efficiency using reporter substrates

  • Monitor effects of SSR2 depletion/overexpression on UPR signaling outputs

  • Assess calcium leakage from ER using fluorescent indicators in conjunction with SSR2 immunostaining

• Tissue-level analysis:

  • Immunohistochemistry in disease models characterized by ER stress (neurodegenerative disorders, diabetes)

  • Multiplex staining of SSR2 with UPR markers and cell-type specific antigens

SSR2 antibodies have been successfully used in detecting expression changes in response to experimental stressors, with polyclonal antibodies providing sensitivity for expression level changes and monoclonal antibodies like clone 31G6 offering precision for localization studies.

What are the experimental approaches for studying SSR2 in different model systems and disease contexts?

Experimental strategies for SSR2 investigation across biological systems:

For disease-specific approaches:

• Cancer research applications:

  • Compare SSR2 expression between tumor and adjacent normal tissues

  • Correlate with ER stress markers (XBP1s, ATF6 cleavage)

  • Examine association with therapy resistance phenotypes

  SSR2 has been implicated in osteosarcoma pathogenesis , suggesting relevance to cancer biology.

• Neurodegenerative disease models:

  • Analyze SSR2 in models of diseases characterized by protein misfolding

  • Co-localization with disease-specific protein aggregates

  • Temporal profiling during disease progression

• Developmental biology:

  • Track SSR2 expression during organogenesis

  • Analyze impact of SSR2 knockout/knockdown on secretory pathway formation

  • Cross-species comparative studies using antibodies with broad reactivity

• Viral infection studies:

  • Examine SSR2 modulation during viral protein overexpression

  • Assess co-localization with viral assembly sites

  • Investigate the role of SSR2 in viral mRNA translation

The polyclonal anti-SSR2 antibody ABIN7270299 offers versatility across multiple species (human, mouse, rat) , making it valuable for comparative studies, while the monoclonal antibody clone 31G6 provides the specificity required for detailed human tissue analysis.

How can researchers quantitatively analyze SSR2 expression data from different experimental approaches?

Quantitative analysis of SSR2 expression requires systematic approaches tailored to specific experimental methods:

• Western blot quantification:

  • Densitometric analysis using ImageJ/FIJI software

  • Normalization strategy: Ratio of SSR2 to housekeeping proteins (β-actin, GAPDH)

  • Statistical approach: Minimum of 3 biological replicates, analyzed by t-test or ANOVA

  • Standard curve generation using recombinant SSR2 protein for absolute quantification

• Immunofluorescence quantification:

  • Cellular compartment analysis: ER-specific signal vs. total cellular fluorescence

  • Z-stack acquisition and 3D reconstruction for volumetric measurement

  • Colocalization analysis: Pearson's or Mander's coefficient with ER markers

  • High-content imaging platforms for population-level statistics

• Flow cytometry analysis:

  • Mean fluorescence intensity (MFI) calculation after gating on viable cells

  • Histogram overlay comparisons between treatment conditions

  • Subpopulation identification based on SSR2 expression levels

  • Multiparameter analysis with UPR markers

• RT-qPCR correlation:

  • Parallel analysis of SSR2 mRNA and protein levels

  • Calculation of protein:mRNA ratios to identify post-transcriptional regulation

  • Time-course studies to determine expression kinetics

• Reproducibility considerations:

  • Antibody lot-to-lot variation control (standard sample inclusion)

  • Instrument calibration with fluorescent standards

  • Blind analysis to prevent investigator bias

Sample data interpretation table:

For proper normalization in quantitative Western blots, researchers should use antibodies validated for linearity across a range of protein concentrations, as demonstrated with the SSR2 antibody H00006746-B01P in human placenta samples .

How can SSR2 antibodies be integrated into multi-omics research approaches?

Integration of SSR2 antibody-based techniques with other omics technologies enables comprehensive understanding of translocon biology:

• Proteomics integration:

  • Immunoprecipitation using SSR2 antibodies followed by mass spectrometry

  • Proximity-dependent biotinylation (BioID or APEX) coupled with SSR2 immunofluorescence

  • Correlation of global proteome changes with SSR2 expression/localization

  • Phospho-proteomic analysis to identify SSR2 post-translational modifications

• Transcriptomics correlation:

  • Single-cell RNA-seq combined with index sorting based on SSR2 protein levels

  • Spatial transcriptomics with SSR2 immunofluorescence on adjacent sections

  • Ribosome profiling to assess translational efficiency of SSR2-dependent substrates

• Structural biology applications:

  • Epitope mapping using hydrogen-deuterium exchange mass spectrometry

  • In situ structural analysis using proximity ligation with structured illumination microscopy

  • Cryo-electron tomography with immunogold labeling using SSR2 antibodies

• Functional genomics approaches:

  • CRISPR screens with SSR2 immunostaining as a phenotypic readout

  • Correlation of genetic variants with SSR2 protein expression in patient-derived samples

  • Synthetic lethality studies in cells with altered SSR2 levels

• Systems biology integration:

  • Network analysis incorporating SSR2 protein interaction data

  • Mathematical modeling of secretory pathway dynamics using quantitative SSR2 data

  • Multi-scale modeling from molecular to cellular levels

The mouse monoclonal antibody against recombinant human SSR2 (clone PAT31G6AT) has shown particular utility in immunoprecipitation applications , making it valuable for proteomics-based interaction studies.

What are the considerations for using SSR2 antibodies in emerging super-resolution microscopy techniques?

Applying SSR2 antibodies in super-resolution microscopy requires specific optimizations:

• STED (Stimulated Emission Depletion) microscopy:

  • Optimal fluorophores: Abberior STAR 580, STAR RED, or Atto 647N

  • Sample preparation: Thinner sections (70-100nm) improve resolution

  • Fixation protocol: 4% PFA followed by 0.1% glutaraldehyde stabilizes structure

  • Expected resolution: 30-50nm resolution of ER membrane microdomains

• STORM/PALM techniques:

  • Fluorophore selection: Alexa Fluor 647, Cy5.5, or photoswitchable fluorescent proteins

  • Buffer system: Oxygen scavenging system with thiol compound (MEA or β-mercaptoethanol)

  • Acquisition parameters: 10,000-30,000 frames for complete reconstruction

  • Drift correction: Fiducial markers (gold nanoparticles) essential for long acquisitions

• Expansion microscopy:

  • Pre-expansion validation: Verify epitope survival after anchoring and digestion

  • Post-expansion staining: May improve antibody access to dense ER regions

  • Expansion factor: 4-10x physical expansion possible with proper protocol optimization

  • Multi-round imaging: SSR2 in first round, followed by other markers

• Lattice light-sheet microscopy:

  • Live-cell compatibility: Consider using anti-SSR2 Fab fragments

  • Phototoxicity minimization: Reduced laser power with sensitive detectors

  • 4D imaging: Capture dynamic SSR2 reorganization during ER stress responses

  • Computational analysis: Specialized tracking algorithms for ER tubule dynamics

• Technical limitations and solutions:

  • Epitope accessibility: Careful permeabilization optimization

  • Label density: Appropriate antibody dilution to achieve Nyquist sampling

  • Signal-to-noise ratio: Background reduction through optimized washing

  • Sample drift: Active focus-locking systems during acquisition

For optimal super-resolution imaging, the directly conjugated anti-SSR2 antibody with DyLight 594 (NBP2-42647DL594) provides advantages of reduced link error compared to secondary antibody detection systems.

What validation standards should researchers apply when evaluating new SSR2 antibodies for specialized applications?

Comprehensive validation of SSR2 antibodies for specialized research applications should follow these guidelines:

• Application-specific validation hierarchy:

  Validation Level	Methodology	Stringency	Application Suitability
  Basic	Western blot with positive control	Minimum	Preliminary studies
  Intermediate	Multiple application testing with controls	Standard	Most research applications
  Advanced	Knockout/knockdown controls, cross-platform	High	Publication-quality research
  Gold standard	Orthogonal validation, epitope mapping	Highest	Critical clinical/research use

• Epitope integrity assessment:

  • Peptide competition assays using the immunizing sequence (e.g., amino acids 18-149 for some antibodies)

  • Cross-reactivity profiling against related proteins (SSR1, SSR3, SSR4)

  • Denaturation sensitivity testing for conformation-dependent epitopes

  • Post-translational modification interference analysis

• Reproducibility standards:

  • Inter-laboratory validation with standardized protocols

  • Consistent results across multiple biological replicates

  • Lot-to-lot comparison with reference standards

  • Benchmarking against established antibodies

• Application-specific benchmarks:

  • Western blot: Single band at expected molecular weight (22kDa for SSR2)

  • Immunofluorescence: Co-localization with established ER markers

  • Flow cytometry: Distinguishable signal between positive and negative populations

  • Immunoprecipitation: Mass spectrometry confirmation of pulled-down protein

• Documentation requirements:

  • Complete experimental conditions and protocols

  • Raw data preservation and accessibility

  • Explicit disclosure of validation limitations

  • Positive and negative control images/data

The scientific community increasingly requires more stringent validation of antibodies. For SSR2 research, antibodies like clone 31G6 that have been validated across multiple platforms (Western blot, ELISA, immunofluorescence) provide greater confidence in experimental results.