SLC35F5 Antibody

What is SLC35F5 and why is it important in research?

SLC35F5 (solute carrier family 35 member F5) is a putative solute transporter membrane protein with a reported length of 523 amino acid residues and a mass of 58.9 kDa in humans . It has gained research interest due to its reported expression in colorectal cancer cells and potential role in cancer biology . As a member of the SLC35F solute transporter protein family, understanding its function and expression patterns provides insights into cellular transport mechanisms and potential disease associations.

The protein has up to 2 reported isoforms and is also known under the synonyms HCV NS5A-transactivated protein 3 and hepatitis C virus NS5A-transactivated protein 3 . SLC35F5 gene orthologs have been identified across multiple species including mouse, rat, bovine, frog, chimpanzee, and chicken, suggesting evolutionary conservation and biological significance .

What are the common applications for SLC35F5 antibodies in research protocols?

SLC35F5 antibodies are primarily employed in the following experimental applications:

When designing experiments, researchers should validate the specific antibody for their application and cell/tissue type, as reactivity and optimal conditions may vary between antibody products .

How do I select the appropriate SLC35F5 antibody for my experimental needs?

Selection should be based on:

• Experimental application: Different antibodies are validated for specific applications (WB, ELISA, IHC, IF). Review validation data from manufacturers for your specific application .

• Species reactivity: Ensure the antibody recognizes your species of interest. Many SLC35F5 antibodies are human-specific, while others show cross-reactivity with mouse, rat, bovine, etc. .

• Clonality: Polyclonal antibodies offer broader epitope recognition but may have batch variation; monoclonal antibodies provide consistency but may be more sensitive to epitope modifications .

• Conjugation needs: Available options include unconjugated, FITC-conjugated, biotin-conjugated, and HRP-conjugated antibodies depending on your detection method .

• Immunogen sequence: Consider whether you need an antibody targeting a specific region (N-terminal, C-terminal, or internal domain) based on your research question .

Methodologically, reviewing existing literature using SLC35F5 antibodies for similar applications can guide selection and experimental design.

What are the recommended validation steps for a new SLC35F5 antibody?

A comprehensive validation protocol should include:

• Western blot analysis: Test with positive control lysates (e.g., colorectal cancer cell lines known to express SLC35F5) to confirm specificity at the expected molecular weight (~58.9 kDa) .

• Peptide competition assay: Pre-incubate antibody with immunizing peptide prior to application to verify specificity.

• Knockout/knockdown controls: Use SLC35F5 knockout or siRNA knockdown samples as negative controls to confirm antibody specificity .

• Cross-reactivity assessment: If using in multi-species studies, validate separately for each species.

• Multiple application testing: If intending to use for different applications (WB, IHC, IF), validate individually for each.

• Concentration optimization: Perform titration experiments to determine optimal working dilutions for each application.

• Signal-to-noise assessment: Compare staining patterns with available literature to ensure expected subcellular localization (membrane) .

Documentation of these validation steps strengthens research reproducibility and reliability of subsequent findings.

How should I optimize immunohistochemistry protocols for SLC35F5 detection in tissue samples?

Optimization for SLC35F5 IHC requires attention to several methodological aspects:

• Fixation protocol: For FFPE tissues, optimal fixation in 10% neutral buffered formalin for 24-48 hours is recommended.

• Antigen retrieval: Test both heat-induced epitope retrieval methods:

  • Citrate buffer (pH 6.0), 95°C for 20 minutes

  • EDTA buffer (pH 9.0), 95°C for 20 minutes

• Blocking optimization: Use 5-10% normal serum from the species of secondary antibody production with 0.1-0.3% Triton X-100 for membrane permeabilization.

• Antibody dilution: Start with manufacturer-recommended dilutions (typically 1:20-1:50 for SLC35F5) and adjust based on signal strength.

• Incubation conditions: Compare overnight incubation at 4°C versus 1-2 hours at room temperature.

• Detection systems: For low expression samples, consider signal amplification methods (e.g., tyramide signal amplification).

• Controls: Include:

  • Positive controls (e.g., colorectal cancer tissue)

  • Negative controls (antibody diluent only)

  • Isotype controls (irrelevant antibody of same isotype)

A systematic approach testing these variables with proper controls will yield optimal staining conditions for reliable SLC35F5 detection.

What considerations are important for western blot analysis of SLC35F5?

For optimal western blot results with SLC35F5 antibodies:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for membrane protein extraction

  • Heat samples at 70°C (not 95°C) for 10 minutes to avoid membrane protein aggregation

• Gel selection:

  • Use 8-10% gels for better resolution of the 58.9 kDa protein

  • Consider gradient gels (4-15%) if analyzing multiple proteins

• Transfer conditions:

  • Use semi-dry or wet transfer with 20% methanol transfer buffer

  • For membrane proteins, longer transfer times (2+ hours) or overnight at lower voltage may improve results

• Blocking optimization:

  • Test both 5% non-fat dry milk and 5% BSA in TBST

  • BSA often works better for phospho-specific antibodies

• Antibody dilution:

  • Follow manufacturer recommendations, typically 0.04-0.4 μg/mL

  • Dilute in the same buffer used for blocking

• Secondary antibody selection:

  • HRP-conjugated, diluted 1:50,000-100,000

  • Consider using secondary antibodies optimized for lower background

• Expected band size:

  • Primary band at ~58.9 kDa

  • Be alert for isoforms or post-translational modifications that may alter migration

• Stripping and reprobing:

  • Mild stripping is recommended if the membrane needs to be reprobed

  • Confirm complete stripping by incubating with secondary antibody alone

Following these methodological considerations will maximize detection specificity and sensitivity when working with SLC35F5 antibodies.

How should I approach SLC35F5 colocalization studies with other membrane proteins?

For high-quality colocalization studies:

• Antibody compatibility:

  • Select SLC35F5 antibodies from different host species than antibodies for other target proteins

  • If same-species antibodies must be used, employ direct conjugation or sequential staining protocols

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider brightness and photostability for detailed imaging

  • Recommended pairs: SLC35F5 with FITC + second target with Cy3/Alexa 594

• Fixation optimization:

  • Test both paraformaldehyde (2-4%) and methanol fixation

  • For membrane proteins, gentle fixation may preserve antigenicity better

• Permeabilization method:

  • Digitonin (25-50 μg/mL) allows selective plasma membrane permeabilization

  • Triton X-100 (0.1%) for complete membrane permeabilization

• Confocal microscopy settings:

  • Use sequential scanning to prevent bleed-through

  • Optimize pinhole size for equivalent optical sections between channels

  • Employ Nyquist sampling criteria for optimal resolution

• Quantitative analysis:

  • Calculate Pearson's correlation coefficient and Mander's overlap coefficient

  • Perform object-based colocalization analysis for punctate structures

  • Use appropriate controls (single stains, competition with blocking peptides)

• Super-resolution approaches:

  • Consider STED, STORM or PALM microscopy for membrane protein colocalization below diffraction limit

This methodological approach enables reliable determination of SLC35F5 spatial relationships with other cellular components.

What are the appropriate approaches for investigating SLC35F5 expression in colorectal cancer research?

Based on recent findings connecting SLC family proteins to colorectal cancer , several specialized approaches are recommended:

• Patient sample selection:

  • Include matched tumor-normal pairs

  • Stratify by cancer stage, grade, and molecular subtypes (MSI status, CMS classification)

  • Consider therapy-naive and post-treatment samples if studying treatment response

• Multi-omics integration:

  • Correlate protein expression (IHC/WB) with transcript levels (qRT-PCR, RNA-seq)

  • Assess methylation status of SLC35F5 promoter regions

  • Consider copy number analysis

• Cell line models:

  • Use appropriate CRC models (HCT116, LOVO) with NCM460 as normal control

  • Verify baseline expression by western blot and qRT-PCR before manipulation

• Expression manipulation strategies:

  • siRNA/shRNA knockdown protocols for loss-of-function studies

  • CRISPR-Cas9 knockout for complete elimination

  • Overexpression models using lentiviral/retroviral systems

• Functional assays:

  • Proliferation (MTT, BrdU incorporation)

  • Migration/invasion (Transwell, wound healing)

  • Colony formation

  • 3D organoid culture phenotyping

• Signaling pathway analysis:

  • Investigate interactions with pathways dysregulated in CRC

  • Consider connection to cAMP signaling pathway based on SLC family research

• Prognostic value assessment:

  • Kaplan-Meier analysis stratifying by SLC35F5 expression

  • Cox regression for multivariate analysis including clinical parameters

Research has shown that other SLC family members like SLC35A2 are upregulated in colorectal cancer and associated with tumor pathological stage and lymph node metastasis, suggesting similar investigations might be valuable for SLC35F5 .

How do I troubleshoot inconsistent or unexpected results when using SLC35F5 antibodies?

When facing technical challenges:

• Non-specific banding in western blots:

  • Increase blocking time/concentration

  • Test alternative blocking agents (milk vs. BSA)

  • Increase washing stringency (higher salt concentration in TBST)

  • Titrate primary antibody concentration

  • Try different antibody targeting alternative epitopes

  • Consider membrane proteins may require specialized extraction methods

• Variable immunostaining intensity:

  • Standardize fixation protocols and times

  • Optimize antigen retrieval conditions

  • Ensure consistent tissue processing and storage

  • Use automated staining platforms for consistency

  • Implement quantitative image analysis methods

• Discrepancies between mRNA and protein expression:

  • Consider post-transcriptional regulation

  • Validate antibody specificity with additional techniques

  • Investigate protein stability and half-life

  • Check for post-translational modifications affecting detection

• Cross-reactivity concerns:

  • Perform peptide competition assays

  • Test antibody on tissues/cells with confirmed SLC35F5 knockout

  • Compare staining patterns with multiple antibodies targeting different epitopes

  • Review potential homology with other SLC family members (e.g., SLC35A2, SLC35F2)

• Technical approach for membrane proteins:

  • Pre-treat with detergents optimized for membrane proteins

  • Consider native vs. denatured detection requirements

  • For flow cytometry, ensure cells remain viable for surface detection

• Differential subcellular localization:

  • Compare with published localization data (nuclear membrane and cytoplasm for SLC35F5)

  • Perform subcellular fractionation to confirm localization biochemically

  • Use counterstains for specific organelles to verify localization

Systematic troubleshooting using these approaches can resolve many technical challenges associated with SLC35F5 antibody applications.

What methods are recommended for studying potential interactions between SLC35F5 and cancer-related pathways?

Based on research connecting SLC family members to oncogenic pathways , consider these methodological approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with SLC35F5 antibodies

  • Proximity ligation assay (PLA) for in situ detection of interactions

  • FRET/BRET analysis for real-time interaction monitoring

  • Mass spectrometry-based interactome analysis

• Pathway analysis techniques:

  • Phosphoproteomic analysis following SLC35F5 manipulation

  • Transcriptomic studies (RNA-seq) after knockdown/overexpression

  • Pharmacological pathway inhibitors combined with SLC35F5 modulation

  • Investigate connections to cAMP signaling pathway identified in SLC research

• Chromatin interactions:

  • ChIP-seq for transcription factors regulating SLC35F5

  • Investigation of epigenetic modifications at the SLC35F5 locus

  • Evaluate connection to HCV NS5A transactivation (suggested by alternative name)

• Cancer-specific contexts:

  • Compare with findings for SLC35A2 in colorectal cancer

  • Investigate potential roles in therapy resistance (noted for SLC35F2)

  • Evaluate immune microenvironment interactions (SLC family members impact immune cell infiltration)

• Functional screening approaches:

  • CRISPR-Cas9 screens in cancer pathway backgrounds

  • Drug sensitivity profiling following SLC35F5 modulation

  • Synthetic lethality screening

These methodologies will help elucidate the functional relationships between SLC35F5 and cancer-associated pathways, potentially revealing new therapeutic opportunities.

How can I investigate the potential role of SLC35F5 in cancer progression and drug resistance mechanisms?

Drawing from related studies on SLC family members :

• Expression correlation studies:

  • Analyze SLC35F5 expression across cancer progression stages

  • Compare primary tumors with matched metastatic samples

  • Evaluate expression changes pre- and post-treatment

• In vitro functional models:

  • Stable knockdown/knockout cell lines to assess:

    • Proliferation/growth rates in 2D and 3D cultures

    • Migration and invasion capabilities

    • Anchorage-independent growth

    • Resistance to apoptosis

• Drug resistance investigations:

  • Drug sensitivity screening before/after SLC35F5 modulation

  • Develop resistant cell lines and assess SLC35F5 expression changes

  • Study potential drug transport functions (as observed with SLC35F2 and YM155)

• Patient-derived models:

  • PDX (patient-derived xenograft) models with varying SLC35F5 expression

  • Patient-derived organoids for ex vivo drug testing

  • Correlation of SLC35F5 expression with treatment outcomes

• Mechanistic studies:

  • Evaluate membrane transport capabilities

  • Investigate impact on cellular metabolites

  • Assess effects on tumor microenvironment

• Clinical correlation approaches:

  • Tissue microarray analysis correlating expression with outcome

  • Create nomograms incorporating SLC35F5 expression with clinical parameters

  • Investigate as potential biomarker for therapy selection

Research on SLC35F2 has shown its involvement in bladder cancer progression and as a potential therapeutic target , suggesting similar approaches may be valuable for SLC35F5 investigation.

What are the methodological challenges in developing selective inhibitors or modulators targeting SLC35F5?

For researchers pursuing therapeutic development:

• Target validation challenges:

  • Confirm SLC35F5's causative role in disease (not merely correlative)

  • Determine if inhibition or activation would be therapeutic

  • Establish minimal functional domains required for activity

• Structural characterization approaches:

  • Membrane protein crystallization challenges

  • Consider cryo-EM for structural determination

  • Homology modeling based on related transporters

  • Molecular dynamics simulations to identify druggable pockets

• Transport activity assays:

  • Develop cell-based transport assays with fluorescent/radioactive substrates

  • Identify physiological substrates through metabolomics

  • Establish high-throughput screening compatible assays

• Compound screening strategies:

  • Fragment-based screening

  • Virtual screening against homology models

  • Repurposing screens of approved drugs

  • Natural product libraries for novel scaffolds

• Selectivity considerations:

  • Cross-screening against related SLC transporters

  • Addressing homology with other SLC35 family members

  • Tissue-specific targeting strategies to minimize off-target effects

• Assessing in vivo activity:

  • Develop appropriate PK/PD models

  • Considerations for blood-brain barrier penetration if relevant

  • Biomarker development for target engagement

• Therapeutic delivery challenges:

  • Strategies for targeting membrane proteins

  • Consideration of antibody-drug conjugates

  • Nanoparticle delivery approaches

The experience with developing inhibitors for SLC35F2 and studying its interaction with the anti-cancer drug YM155 provides a potential blueprint for similar approaches with SLC35F5.

How can I improve detection sensitivity for low-abundance SLC35F5 in clinical samples?

For enhanced detection:

• Sample preparation optimization:

  • Enrich for membrane fractions before analysis

  • Use phosphatase/protease inhibitors to prevent degradation

  • Consider specific membrane protein extraction buffers containing appropriate detergents

• Signal amplification methods:

  • For IHC: Tyramide signal amplification (TSA)

  • For western blot: Enhanced chemiluminescence (ECL) substrates with extended exposure

  • For IF: Quantum dots or multi-layer detection systems

• Antibody concentration:

  • Optimize primary antibody concentration through careful titration

  • Extended incubation times at 4°C (overnight for IHC/IF, 24-48h for WB)

• Alternative detection strategies:

  • Proximity ligation assay (PLA) for single molecule detection

  • RNAscope for parallel mRNA visualization

  • Mass spectrometry with targeted peptide detection

• Preprocessing methods:

  • Antigen retrieval optimization (test multiple buffers and pH conditions)

  • Evaluate different fixatives for tissue preservation

  • Consider non-formalin fixatives for membrane protein preservation

• Technical handling:

  • Minimize freeze-thaw cycles for antibodies

  • Use fresh tissue samples when possible

  • Implement standardized protocols with minimal variations

These approaches can significantly improve detection of challenging or low-abundance SLC35F5 expression in clinical specimens.

What are the best practices for quantitative analysis of SLC35F5 expression in cancer specimens?

For reliable quantitation:

• Standardization procedures:

  • Include calibration standards on each blot/slide

  • Process all comparative samples simultaneously

  • Use internal loading controls appropriate for your sample type

• Image acquisition protocols:

  • Standardize exposure settings for all samples

  • Ensure linear dynamic range for quantification

  • Use calibrated imaging systems

• Quantification methods for IHC:

  • H-score methodology (intensity × percentage positive cells)

  • Digital image analysis with validated algorithms

  • Consider both intensity and localization patterns

  • Reference research methodology used for SLC35A2 in CRC

• Western blot quantification:

  • Densitometric analysis normalized to appropriate loading controls

  • Use standard curves with recombinant protein if absolute quantification needed

  • Verify linear range of detection for your system

• Multi-method validation:

  • Correlate protein levels (WB/IHC) with mRNA expression (qRT-PCR)

  • Consider proteomics approaches for absolute quantification

  • Implement tissue microarrays for high-throughput analysis

• Statistical considerations:

  • Power calculation to determine appropriate sample sizes

  • Non-parametric methods for non-normally distributed data

  • Multivariate analysis to account for covariates

• Reporting standards:

  • Document all methodological details for reproducibility

  • Include representative images showing scoring categories

  • Report antibody validation methods and controls

Following these practices will enhance reliability and reproducibility of quantitative SLC35F5 expression data.

How should I design experiments to differentiate between SLC35F5 and other closely related SLC family members?

To ensure specificity:

• Sequence analysis preparation:

  • Perform sequence alignment of SLC35F5 with related family members

  • Identify regions of high homology versus unique sequences

  • Select antibodies targeting unique epitopes when possible

• Transcript-level discrimination:

  • Design PCR primers spanning unique exon junctions

  • Implement specific probe-based qPCR assays

  • Consider RNAscope for in situ discrimination of related transcripts

• Protein-level specificity:

  • Use multiple antibodies targeting different epitopes

  • Perform peptide competition assays with specific peptides

  • Consider western blotting with size discrimination (SLC35F5: 58.9 kDa)

• Knockout/knockdown validation:

  • Generate specific SLC35F5 knockdown and measure cross-reactivity

  • Use CRISPR-Cas9 gene editing for complete elimination

  • Test antibody specificity in knockout model systems

• Mass spectrometry approaches:

  • Targeted proteomics with peptide-specific transitions

  • Parallel reaction monitoring (PRM) for specific detection

  • Identification of unique post-translational modifications

• Functional discrimination:

  • Develop transport assays specific to SLC35F5 substrates

  • Compare phenotypic outcomes of specific family member modulation

  • Evaluate differential subcellular localization

• Comparative expression analysis:

  • Document tissue-specific expression patterns

  • Compare regulation under various conditions

  • Examine differences in cancer type associations

These approaches will help distinguish SLC35F5 from related transporters like SLC35A2 and SLC35F2 , which have established roles in colorectal and bladder cancers, respectively.