SMIM19 Antibody

What is SMIM19 and why is it important to study?

SMIM19, also known as C8orf40 or UPF0697 protein C8orf40, is a small integral membrane protein encoded by the SMIM19 gene. While its precise biological function remains under investigation, it's part of the broader family of small integral membrane proteins that often play crucial roles in cellular signaling, membrane organization, and protein-protein interactions. Research into SMIM19 contributes to our understanding of fundamental cellular processes and potential disease associations. Current commercially available antibodies are primarily directed against human SMIM19, with highest sequence identity to mouse (80%) and rat (77%) orthologs .

What types of SMIM19 antibodies are currently available for research?

As of early 2025, researchers have access to multiple SMIM19 antibody options. The primary types include:

Both available antibodies are rabbit polyclonal antibodies targeting human SMIM19, though with different concentration formats and validation profiles .

Which experimental applications are SMIM19 antibodies validated for?

Current commercially available SMIM19 antibodies have been validated for several standard immunological applications:

• Immunohistochemistry (IHC): For detection of SMIM19 in fixed tissue sections

• Western Blot (WB): For detection of denatured SMIM19 in protein lysates

• Immunocytochemistry/Immunofluorescence (ICC-IF): For cellular localization studies

These validations ensure researchers can reliably employ these antibodies across multiple experimental platforms depending on their specific research questions .

How does epitope selection influence SMIM19 antibody performance?

The performance of SMIM19 antibodies is significantly influenced by epitope selection. The Invitrogen SMIM19 polyclonal antibody, for example, was generated using a specific immunogen sequence: "KRNKRRIMRI FSVPPTEETL SEPNFYDTIS KIRLRQQLEM YSISRKYDYQ QPQNQADSVQ LSLE" . This sequence selection impacts:

• Cross-reactivity with orthologs (80% identity with mouse, 77% with rat)

• Accessibility of the epitope in different applications

• Potential interference with protein-protein interactions

• Antibody performance in various buffer conditions

When designing experiments, researchers should consider whether their application requires detection of specific domains or regions of SMIM19, particularly when investigating membrane topology or protein interactions.

What considerations are important when validating SMIM19 antibody specificity?

Validating antibody specificity is crucial for SMIM19 research, particularly given its membrane protein nature and potential for cross-reactivity. Key validation approaches include:

• Negative controls: Testing in systems where SMIM19 is knocked down or knocked out

• Orthogonal detection methods: Comparing antibody results with tagged SMIM19 expression

• Cross-reactivity assessment: Testing in tissues/cells from different species to validate ortholog detection claims

• Multiple antibody comparison: Using antibodies targeting different SMIM19 epitopes to confirm detection patterns

Additionally, validation should be application-specific, as an antibody performing well in Western blot may not necessarily work in immunohistochemistry due to differences in protein conformation and epitope accessibility .

What are the critical parameters for optimizing SMIM19 detection in membrane protein preparations?

As a small integral membrane protein, SMIM19 presents unique challenges for detection. Optimization considerations include:

• Extraction methodology: Selection of detergents that efficiently solubilize membrane proteins while preserving epitope structure

• Sample preparation: Temperature conditions during denaturation can affect aggregation

• Blocking optimization: Membrane proteins often require specialized blocking conditions to reduce background

• Incubation parameters: Extended primary antibody incubation times may be necessary for optimal detection

• Detection system selection: Signal amplification methods for low-abundance membrane proteins

Researchers working with SMIM19 should consider starting with established membrane protein protocols and then systematically optimizing conditions for their specific experimental system and antibody .

What is the recommended protocol for SMIM19 detection in Western blotting?

For optimal Western blot detection of SMIM19, consider the following methodological approach:

• Sample preparation:

  • Use lysis buffers containing 1-2% non-ionic detergents (e.g., Triton X-100, NP-40)

  • Include protease inhibitor cocktails to prevent degradation

  • Heat samples at 70°C instead of 95°C to reduce membrane protein aggregation

• Gel selection and transfer:

  • Use gradient gels (4-20%) to better resolve small membrane proteins

  • Transfer at lower voltage for extended time (25V overnight) to improve transfer efficiency

• Antibody protocol:

  • Block with 5% BSA in TBST rather than milk proteins

  • Dilute primary antibody appropriately (1:500 for Atlas antibody at 0.05 mg/ml; 1:1000-1:2000 for Invitrogen antibody at 0.2 mg/ml)

  • Incubate overnight at 4°C to maximize signal

  • Use secondary antibodies specifically validated for membrane protein work

• Detection considerations:

  • Enhanced chemiluminescence systems offer good sensitivity for SMIM19 detection

  • Consider longer exposure times to detect low-abundance signal

How should researchers approach SMIM19 immunohistochemistry optimization?

Optimizing SMIM19 detection in tissue sections requires careful attention to fixation and antigen retrieval:

• Fixation considerations:

  • Formalin-fixed paraffin-embedded (FFPE) tissues require appropriate antigen retrieval

  • Fresh-frozen sections may better preserve membrane protein epitopes

• Antigen retrieval options:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Alternative retrieval using EDTA buffer (pH 9.0) if citrate proves insufficient

• Blocking and antibody incubation:

  • Extended blocking (1-2 hours) with serum-free blockers to reduce background

  • Primary antibody dilutions starting at 1:100 for initial optimization

  • Extended incubation times (overnight at 4°C) often improve signal quality

• Detection systems:

  • Polymer-based detection systems often provide better results than biotin-streptavidin

  • Tyramide signal amplification for enhanced sensitivity in low-expression tissues

What strategies can address common troubleshooting issues with SMIM19 antibodies?

When encountering problems with SMIM19 antibody performance, consider these targeted troubleshooting strategies:

• High background issues:

  • Increase blocking time and concentration

  • Perform additional washing steps with increased detergent (0.1-0.3% Tween-20)

  • Reduce primary and secondary antibody concentrations

  • Consider using alternative blocking agents (casein, fish gelatin)

• Weak or no signal detection:

  • Verify protein expression in your experimental system

  • Optimize protein extraction for membrane proteins

  • Test different antigen retrieval methods for IHC

  • Increase antibody concentration and incubation time

• Non-specific bands in Western blot:

  • Increase washing stringency

  • Optimize primary antibody dilution

  • Consider alternative blocking reagents

  • Validate with additional negative controls

How can researchers effectively integrate SMIM19 antibodies into co-localization studies?

Co-localization studies with SMIM19 require careful planning around antibody compatibility:

• Primary antibody selection:

  • Choose SMIM19 antibodies raised in different host species than other target antibodies

  • Consider using directly conjugated antibodies to avoid cross-reactivity

• Imaging optimization:

  • Begin with single-channel controls to establish detection parameters

  • Use appropriate filter sets to minimize spectral overlap

  • Employ sequential scanning in confocal microscopy

• Quantitative co-localization:

  • Use established coefficients (Pearson's, Manders') for quantification

  • Establish thresholds using appropriate controls

  • Analyze multiple fields and biological replicates

• Validation approaches:

  • Complement antibody detection with genetically tagged constructs

  • Use super-resolution techniques for detailed co-localization analysis

What controls should be incorporated when studying SMIM19 expression patterns?

Robust controls are essential for meaningful SMIM19 expression studies:

• Negative controls:

  • Isotype controls to assess non-specific binding

  • Secondary-only controls to evaluate background

  • SMIM19 knockdown/knockout samples when available

• Positive controls:

  • Tissues/cells with confirmed SMIM19 expression

  • Recombinant SMIM19 protein as Western blot standard

  • Over-expression systems for antibody validation

• Orthogonal validation:

  • Correlate protein detection with mRNA expression data

  • Use multiple antibodies targeting different SMIM19 epitopes

  • Complement immunological detection with mass spectrometry when possible

How does SMIM19 antibody selection compare to antibody approaches for other membrane proteins?

When comparing SMIM19 antibody work to other membrane protein research, consider these distinctive aspects:

• Size considerations:

  • As a small integral membrane protein, SMIM19 may require specialized extraction methods

  • Gel systems may need optimization compared to larger membrane proteins

• Epitope accessibility:

  • Membrane topology affects epitope exposure differently than for multi-pass transmembrane proteins

  • Native conformation preservation may be more crucial for certain applications

• Cross-reactivity challenges:

  • Sequence conservation with orthologs (80% mouse, 77% rat) informs cross-species applications

  • Alternative splicing or processing variants may affect detection

• Technical approaches:

  • Immunoprecipitation optimization differs from soluble proteins

  • Fixation and permeabilization requirements may be distinct

How might advanced antibody technologies enhance SMIM19 research?

Emerging antibody technologies offer promising approaches for advancing SMIM19 research:

• Recombinant antibody development:

  • Single-chain variable fragments (scFvs) for improved membrane protein access

  • Nanobodies/single-domain antibodies for enhanced epitope accessibility

  • Site-specific conjugation for improved imaging applications

• Proximity labeling approaches:

  • Antibody-enzyme fusions for proximity-dependent labeling

  • Identification of SMIM19 interaction partners in native contexts

  • Spatial mapping of SMIM19 within membrane microdomains

• Conformation-specific antibodies:

  • Development of antibodies specific to different SMIM19 conformational states

  • Tools for studying structural dynamics in different cellular contexts

What methodological innovations could address current limitations in SMIM19 antibody applications?

Several methodological innovations could overcome current SMIM19 antibody limitations:

• Membrane protein-specific protocols:

  • Improved extraction methods preserving native membrane protein complexes

  • Enhanced in situ detection without membrane disruption

  • Native membrane protein separation techniques

• Quantitative approaches:

  • Absolute quantification standards for SMIM19 detection

  • Multiplexed detection systems for co-expression analysis

  • Automated image analysis workflows for expression pattern recognition

• Single-cell applications:

  • Adaptation of SMIM19 antibodies for single-cell proteomics

  • Integration with single-cell transcriptomics data

  • Spatial analysis of SMIM19 distribution at subcellular resolution