MFSD14B Antibody

What is MFSD14B and why is it of interest in neuroscience research?

MFSD14B is a 506-amino acid protein with approximately 12 predicted transmembrane regions, characteristic of the MFS transporter family. This protein is expressed throughout the mouse brain, with particularly notable expression in neurons where it localizes to the endoplasmic reticulum (ER), as evidenced by co-localization with the KDEL ER retention marker . The protein's high conservation between mouse and human (93.8% similarity) suggests important biological functions .

MFSD14B is of particular interest because:

• It shows differential expression in response to metabolic challenges such as amino acid starvation and high-fat diet

• Its expression changes specifically in certain brain regions (upregulated in striatum, downregulated in hypothalamus and brainstem during high-fat diet)

• It has a distinct intracellular localization pattern suggesting specific subcellular functions

• Its phylogenetic relationship with other MFS transporters suggests potential roles in substrate transport, possibly related to energy metabolism

While its precise function remains unknown, these characteristics make MFSD14B antibodies valuable tools for investigating neuronal metabolism and stress responses.

What verification methods should I use to confirm MFSD14B antibody specificity?

To verify MFSD14B antibody specificity, implement these methodological approaches:

• Western blot validation:

  • Expected band size: Approximately 55-59 kDa (the slight discrepancy from the predicted 55 kDa may be due to post-translational modifications)

  • Include positive control tissues: Brain tissue and skeletal muscle show high expression

  • Include negative controls: Consider tissues with minimal expression or knockout/knockdown samples

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Perform parallel immunostaining/Western blot with blocked and unblocked antibody

  • Signal elimination in the blocked sample confirms specificity

• Cross-reactivity testing:

  • Test for potential cross-reactivity with MFSD14A, which shares 75.4% sequence similarity (85.6% in protein domains)

  • Use MFSD14A-expressing vs. MFSD14B-expressing constructs as controls

• Multiple antibody validation:

  • Compare staining patterns using antibodies targeting different epitopes of MFSD14B

  • Consistent localization patterns increase confidence in specificity

• siRNA knockdown validation:

  • Confirm signal reduction in samples with reduced MFSD14B expression

What subcellular localization pattern should I expect when using MFSD14B antibodies?

When using MFSD14B antibodies for subcellular localization studies, expect:

• Primary localization: Endoplasmic reticulum - MFSD14B shows strong co-localization with the KDEL ER retention marker in primary mouse embryonic cortex cultures

• Cell type specificity: Predominantly neuronal expression - MFSD14B co-localizes with neuronal markers (NeuN and Pan neuronal marker) but not with astrocyte markers (GFAP)

• Staining pattern:

  • Diffuse intracellular pattern throughout the cell body

  • Not localized to the plasma membrane (no spatial overlap with Pan neuronal antibody at cell boundaries)

  • More spread out staining pattern compared to the Golgi-localized MFSD14A

• Regional distribution:

  • Present throughout the brain but with a scattered pattern and lower density compared to MFSD14A

  • Notable staining in cortex, striatum, hippocampus, around the third ventricle and hypothalamus

  • Prominent staining in cerebellar lobules and facial nucleus

• Visualization notes:

  • Both cell bodies and neuronal projections can be visualized

  • Not all neurons (Pan-positive cells) express MFSD14B, suggesting cell-type specific expression patterns

How should I optimize fixation methods for MFSD14B immunohistochemistry?

Optimal fixation methods for MFSD14B immunohistochemistry should consider its transmembrane nature and ER localization:

For brain tissue sections:

• Paraformaldehyde fixation protocol:

  • 4% PFA perfusion followed by post-fixation (4-24 hours)

  • This method preserves morphology while maintaining antibody accessibility to MFSD14B epitopes

  • Compatible with both fluorescent and non-fluorescent detection methods

• Section thickness considerations:

  • For overview studies: 70 μm free-floating sections work well for non-fluorescent immunohistochemistry

  • For co-localization studies: 5-10 μm paraffin sections allow better resolution for double-labeling

For primary neuronal cultures:

• Fixation for subcellular localization:

  • Brief fixation (10-15 minutes) with 4% PFA at room temperature

  • PBS washing to remove excess fixative

  • This approach preserves the intracellular architecture while allowing antibody penetration

• Membrane permeabilization:

  • Since MFSD14B is an intracellular protein, adequate permeabilization is crucial

  • 0.1-0.3% Triton X-100 for 10-15 minutes enhances antibody access to ER structures

Important considerations:

• Overfixation may mask epitopes, particularly for transmembrane proteins

• For double-labeling studies with intracellular markers like KDEL, optimize permeabilization to maintain organelle structure while allowing antibody access

• Super-resolution microscopy techniques (like ELYRA) may be necessary to resolve the detailed subcellular localization pattern

What experimental controls are essential when using MFSD14B antibodies?

When designing experiments with MFSD14B antibodies, incorporate these essential controls:

For Western blotting:

• Positive tissue controls: Include brain tissue and skeletal muscle samples which show high expression of MFSD14B

• Molecular weight markers: Critical for confirming the expected ~59 kDa band (note that membrane proteins may migrate differently than predicted)

• Loading controls: Use housekeeping proteins appropriate for the subcellular fraction being examined (ER-specific controls may be more relevant than general cytoplasmic markers)

For immunohistochemistry/immunocytochemistry:

• Primary antibody omission: To assess non-specific binding of secondary antibodies

• Isotype control: Using matched isotype IgG at the same concentration to identify non-specific binding

• Competing peptide control: Pre-absorption with immunizing peptide to demonstrate specificity

• Positive controls: Include tissues known to express MFSD14B (cortex, striatum, cerebellum)

For co-localization studies:

• Single-labeled controls: Essential for determining bleed-through in fluorescent channels

• Known marker co-staining: Include established markers such as:

  • KDEL for ER localization confirmation

  • NeuN for neuronal identification

  • GFAP to confirm lack of astrocytic expression

For expression studies:

• Reference gene validation: When studying MFSD14B expression changes, validate stable reference genes under your experimental conditions

• Time-course controls: Important when studying starvation responses, as MFSD14B expression changes over time even in control conditions

How can I design experiments to investigate MFSD14B regulation during metabolic stress?

Based on research showing MFSD14B expression changes during amino acid starvation and high-fat diet, consider these methodological approaches:

Experimental design for amino acid starvation studies:

• Cell culture model:

  • Primary embryonic cortex cultures (E14-16) have shown robust MFSD14B regulation

  • Timeline: Include early (3h), intermediate (7h), and extended (12h) timepoints to capture both acute and adaptive responses

  • Controls: Include time-matched controls with complete media, as MFSD14B expression changes over time even under normal conditions

• Quantification approaches:

  • qRT-PCR for transcript analysis (see primer design considerations below)

  • Western blot for protein level changes (account for potential post-translational modifications)

  • Immunofluorescence for localization changes during stress

• Data analysis considerations:

  • Express results as percentage of the highest expression sample rather than comparing only within timepoints

  • Statistical analysis: Paired comparisons between control and treatment at each timepoint

  • Consider correlation with known stress response genes (ATF4, CHOP, BiP)

High-fat diet experimental framework:

• In vivo model parameters:

  • Diet duration: 8 weeks shows significant effects on MFSD14B expression

  • Weight gain monitoring: Significant weight difference (38% ± 9% for HFD vs. 12% ± 2.3% for controls) correlates with expression changes

• Region-specific analysis:

  • Focus on striatum (upregulation), hypothalamus and brainstem (downregulation)

  • Include cerebellum and cortex as potentially non-responsive control regions

• Comprehensive approach:

  • Combine transcript quantification with protein analysis and immunohistochemistry

  • Consider cell type-specific changes using double-labeling techniques

  • Correlate with metabolic parameters (glucose, insulin, leptin levels)

Table 1: Observed MFSD14B Expression Changes in Metabolic Stress Models

What approaches can distinguish between MFSD14A and MFSD14B in research applications?

Antibody-based discrimination strategies:

• Epitope selection for antibody development:

  • Target regions with lowest sequence conservation between MFSD14A and MFSD14B

  • Consider N- or C-terminal domains which typically have higher divergence in MFS transporters

  • Validate antibody specificity against recombinant proteins of both transporters

• Cross-reactivity testing protocol:

  • Express tagged versions of MFSD14A and MFSD14B in cell lines

  • Perform parallel Western blots with anti-tag antibodies and specific antibodies

  • Compare immunofluorescence patterns with known localizations (MFSD14A in Golgi, MFSD14B in ER)

Subcellular localization as a distinguishing factor:

• Co-localization panel design:

  • MFSD14A: Include Golgi markers like Giantin

  • MFSD14B: Include ER markers like KDEL

  • Apply super-resolution microscopy for definitive localization

• Differential fractionation approach:

  • Perform subcellular fractionation to separate Golgi and ER fractions

  • Compare enrichment patterns of MFSD14A vs MFSD14B by Western blot

Molecular biology approaches for specific detection:

• Primer design for discriminative qPCR:

  • Design primers in divergent regions (3' UTR is often optimal)

  • Validate specificity using plasmids containing each transporter

  • Include melt curve analysis to confirm amplification of single products

• RNA interference specificity:

  • Design siRNAs targeting unique regions of each transcript

  • Validate selective knockdown using both qPCR and Western blot

  • Monitor potential compensatory expression changes

Table 2: Distinguishing Features Between MFSD14A and MFSD14B

How can I optimize Western blotting protocols specifically for MFSD14B detection?

Detecting MFSD14B by Western blot requires optimization due to its transmembrane nature and post-translational modifications:

Sample preparation considerations:

• Membrane protein extraction:

  • Use specialized membrane protein extraction buffers containing mild detergents (RIPA buffer with 0.1% SDS)

  • Avoid excessive heating which can cause membrane protein aggregation

  • Consider using specialized membrane protein solubilization buffers for enhanced extraction

• Glycoprotein handling:

  • MFSD14B has 13 predicted O-GalNAc glycosylation sites

  • For deglycosylation studies, treat samples with appropriate glycosidases

  • Enzymatic deglycosylation can confirm the contribution of glycosylation to the observed band size difference (59 kDa vs. predicted 55 kDa)

Electrophoresis and transfer optimizations:

• SDS-PAGE parameters:

  • Use 10-12% acrylamide gels for optimal resolution in the 50-60 kDa range

  • Lower temperatures during electrophoresis (4°C) can improve membrane protein resolution

  • Consider specialized gel systems designed for membrane proteins

• Transfer considerations:

  • Optimize transfer time and voltage for proteins in the 55-60 kDa range

  • Add SDS (0.01-0.02%) to transfer buffer to facilitate movement of hydrophobic proteins

  • Consider semi-dry transfer systems for more efficient transfer of transmembrane proteins

Detection refinements:

• Blocking optimization:

  • Test BSA vs. milk-based blocking (milk proteins can sometimes interact with antibodies against membrane proteins)

  • Consider specialized blocking reagents for membrane proteins

  • Optimize blocking time to minimize background while preserving specific signal

• Signal interpretation guidance:

  • Expected band at approximately 59 kDa

  • Possible detection of multiple bands due to different glycosylation states

  • Potential detection of degradation products if the antibody recognizes fragments

Additional validation approaches:

• Parallel detection strategy:

  • Compare results using antibodies against different epitopes

  • Include genetic models (overexpression, knockdown) to validate band identity

• Subcellular fractionation verification:

  • Confirm enrichment in ER membrane fractions

  • Compare distribution pattern with known ER markers

Table 3: Troubleshooting MFSD14B Western Blot Issues

What experimental approaches can elucidate MFSD14B's potential substrates and transport function?

Although MFSD14B's substrates and transport functions remain unknown, these methodological approaches can help elucidate its role:

Phylogenetic analysis-guided hypothesis development:

• Computational approaches:

  • Phylogenetic clustering analysis places MFSD14B near SLC families 15, 19, 22, 29, and 43

  • Use sequence similarity and structural predictions to narrow potential substrate classes

  • Molecular modeling of transmembrane domains to identify potential substrate binding sites

• Transport assay design based on related transporters:

  • Test substrates of phylogenetically related SLCs (oligopeptides, amino acids, nucleosides)

  • Design radiolabeled substrate uptake studies in overexpression systems

  • Consider electrophysiological approaches if ion coupling is suspected

ER localization-specific functional investigations:

• ER luminal substrate measurement techniques:

  • Develop targeted sensors for potential substrates in the ER lumen

  • Apply subcellular fractionation to measure substrate concentrations in ER vesicles

  • Consider ER-targeted metabolomic approaches comparing wild-type and knockdown models

• ER stress connection assessment:

  • Evaluate MFSD14B's role in the unfolded protein response pathway

  • Measure ER stress markers in MFSD14B knockdown/knockout models

  • Test if MFSD14B expression correlates with various ER stress inducers

Metabolic perturbation studies:

• Starvation response experimental framework:

  • Design amino acid deprivation experiments with metabolomics analysis

  • Focus on early timepoints (3h) when MFSD14B expression significantly increases

  • Profile changes in specific metabolite classes in control vs. MFSD14B-deficient cells

• High-fat diet correlation analysis:

  • Correlate region-specific MFSD14B expression changes (up in striatum, down in hypothalamus/brainstem) with metabolite profiles

  • Perform targeted metabolomics on relevant tissue regions

  • Test if MFSD14B knockdown prevents metabolic adaptations to high-fat diet

Interaction partner identification:

• Proximity labeling approaches:

  • Apply BioID or APEX2 proximity labeling techniques with MFSD14B as the bait

  • Focus on ER-resident interaction partners

  • Validate interactions through co-immunoprecipitation and functional studies

• Genetic screens for functional partners:

  • Perform synthetic lethality screens in cellular models

  • Use CRISPR interference screens under metabolic stress conditions

  • Identify genes with expression profiles that correlate with MFSD14B across tissues

How can I effectively use MFSD14B antibodies in co-localization studies with other cellular markers?

For effective co-localization studies of MFSD14B with other cellular markers, implement these methodological approaches:

Optimizing multi-labeling protocols:

• Sequential immunostaining approach:

  • Begin with the weakest signal (typically MFSD14B) for maximum sensitivity

  • Use directly conjugated secondary antibodies to minimize cross-reactivity

  • Consider tyramide signal amplification for detecting low abundance proteins

• Antibody selection strategies:

  • Choose primary antibodies from different host species to avoid cross-reactivity

  • For same-species antibodies, use directly conjugated primaries or specialized detection kits

  • Validate each antibody individually before combining for co-localization

ER markers for definitive co-localization:

• Recommended ER marker panel:

  • KDEL (retention signal) – confirmed co-localization with MFSD14B

  • Calnexin (ER membrane protein)

  • Sec61 (translocon complex)

  • BiP/GRP78 (ER lumen chaperone)

• Experimental approach:

  • Super-resolution microscopy (e.g., ELYRA as used in primary research )

  • Z-stack imaging to capture the full spatial distribution

  • Quantitative co-localization analysis using Pearson's or Mander's coefficients

Neuronal subtype marker co-localization:

• Verified neuronal markers:

  • NeuN – confirmed co-localization with MFSD14B in brain sections

  • Pan neuronal marker – confirmed in primary cultures

  • Add specific neuronal subtype markers (e.g., GABAergic, glutamatergic) to determine cell-type specificity

• Technical considerations:

  • Use thin sections (5-10 μm) for optimal resolution in brain tissue

  • Primary cultures provide better subcellular resolution than tissue sections

  • Include z-stack analysis to distinguish true co-localization from signal overlap

Stress response pathway markers:

• Metabolic stress markers:

  • ER stress sensors (IRE1α, PERK, ATF6)

  • Autophagy markers (LC3, p62)

  • Nutrient sensing pathway components (mTOR, AMPK)

• Co-localization during dynamic responses:

  • Time-course experiments during amino acid starvation

  • Fixed timepoints during high-fat diet studies

  • Correlation of localization changes with expression level changes

Image acquisition and analysis guidance:

• Acquisition parameters:

  • Collect sequential images to prevent bleed-through

  • Maintain consistent exposure settings across experimental conditions

  • Include single-labeled controls for each fluorophore

• Quantification methods:

  • Pixel intensity correlation analysis for co-localization degree

  • Object-based co-localization for discrete structures

  • Line scan analysis across subcellular compartments

Table 4: Recommended Marker Combinations for MFSD14B Co-localization Studies

How can MFSD14B antibodies be applied in studies of metabolic disorders?

Given MFSD14B's differential regulation during metabolic challenges, these methodological approaches can be applied in metabolic disorder research:

Obesity and high-fat diet studies:

• Region-specific analysis framework:

  • Focus on striatum (upregulation), hypothalamus and brainstem (downregulation) during high-fat diet

  • Compare expression patterns across different obesity models (genetic vs. diet-induced)

  • Correlate MFSD14B levels with metabolic parameters and feeding behaviors

• Cellular response assessment:

  • Evaluate neuron-specific responses through immunohistochemistry

  • Quantify MFSD14B changes at both transcript and protein levels

  • Investigate potential compensatory roles of MFSD14A in MFSD14B-deficient models

Starvation and nutrient sensing investigations:

• Temporal dynamics approach:

  • Focus on early response (3h) when MFSD14B is significantly upregulated during amino acid starvation

  • Design recovery experiments to determine reversibility of expression changes

  • Compare with other nutrient stress responses (glucose deprivation, lipid depletion)

• Signaling pathway integration:

  • Investigate connections to mTOR pathway components

  • Test involvement in AMPK-mediated responses

  • Evaluate potential roles in amino acid sensing pathways

ER stress in metabolic disease:

• Analytical approach:

  • Co-localization with ER stress markers in metabolic disease models

  • Quantitative assessment of MFSD14B changes during ER stress induction

  • Comparison between peripheral and central nervous system responses

• Intervention studies:

  • Test if MFSD14B modulation affects ER stress outcomes

  • Evaluate pharmacological ER stress modulators on MFSD14B expression

  • Correlate therapeutic responses with MFSD14B expression changes

Translational considerations:

• Human tissue analysis:

  • Leverage the high conservation between mouse and human MFSD14B (93.8% similarity)

  • Validate antibody specificity in human samples

  • Compare expression patterns in control vs. metabolic disorder samples

• Biomarker potential assessment:

  • Evaluate MFSD14B detection in accessible samples (CSF, blood cells)

  • Correlate expression changes with disease progression

  • Test sensitivity to therapeutic interventions

What methodological considerations are important when generating or selecting MFSD14B antibodies for specialized applications?

When generating or selecting MFSD14B antibodies for specialized research applications, consider these methodological aspects:

Epitope selection strategies:

• Structural considerations:

  • Avoid transmembrane domains which have poor immunogenicity

  • Target extramembranous loops, particularly those facing the ER lumen

  • Consider the 13 predicted O-GalNAc glycosylation sites which may mask epitopes

• Specificity engineering:

  • Select regions with minimal homology to MFSD14A

  • Avoid conserved MFS motifs that might cross-react with other transporters

  • Consider species conservation if cross-reactivity between models is desired

Application-specific antibody selection:

• For Western blotting:

  • Linear epitopes that survive denaturation

  • N- or C-terminal targeting often yields cleaner results

  • Consider detecting both glycosylated and non-glycosylated forms

• For immunohistochemistry/immunocytochemistry:

  • Conformational epitopes may provide higher specificity

  • Test fixation compatibility extensively

  • Validate penetration into ER structures

• For immunoprecipitation:

  • High-affinity antibodies are essential

  • Test under native conditions to preserve protein-protein interactions

  • Consider tag-based approaches for difficult-to-immunoprecipitate proteins

Production and purification considerations:

• Monoclonal versus polyclonal selection:

  • Monoclonals: Higher specificity, lower batch variation, potential epitope limitation

  • Polyclonals: Multiple epitope recognition, higher sensitivity, batch variability concerns

• Validation requirements:

  • Knockout/knockdown controls

  • Peptide competition assays

  • Cross-reactivity testing with MFSD14A

  • Testing across multiple applications

Specialized application considerations:

• For super-resolution microscopy:

  • Bright, photostable fluorophore conjugates

  • Minimal background staining

  • High signal-to-noise ratio

• For multiplex imaging:

  • Compatibility with multiple labeling protocols

  • Available in diverse host species

  • Directly conjugated options

• For FACS applications:

  • Surface-accessible epitopes if examining permeabilized cells

  • High sensitivity for detection of low-abundance proteins

  • Low non-specific binding to prevent false positives

Table 5: Strategic Approaches for MFSD14B Antibody Development

How might MFSD14B antibodies contribute to understanding the transporter's role in neurodegenerative conditions?

While direct links between MFSD14B and neurodegenerative conditions are not yet established, its ER localization and responsiveness to metabolic stress suggest potential relevance. Consider these methodological approaches:

ER stress connection investigations:

• Experimental framework:

  • Examine MFSD14B expression in models of neurodegenerative diseases with prominent ER stress components (Alzheimer's, Parkinson's, ALS)

  • Analyze co-localization with disease-specific protein aggregates

  • Test if MFSD14B modulation affects disease protein handling

• Technical approach:

  • Multiplex immunofluorescence with MFSD14B and disease markers

  • Quantitative analysis of expression changes during disease progression

  • Region-specific analysis focusing on vulnerable neuronal populations

Metabolic dysfunction exploration:

• Investigative strategy:

  • Given MFSD14B's response to metabolic challenges , examine its role in the metabolic components of neurodegeneration

  • Test correlation between MFSD14B expression and regional metabolic deficits

  • Investigate modulation of MFSD14B as a potential compensatory mechanism

• Methodological considerations:

  • Combine PET imaging of metabolic activity with post-mortem MFSD14B analysis

  • Use neuron-specific conditional knockdown models

  • Apply metabolomic profiling in affected brain regions

Therapeutic targeting potential:

• Screening approaches:

  • Develop high-content screening assays using MFSD14B antibodies

  • Test compounds that modulate MFSD14B expression or function

  • Focus on restoration of normal expression patterns in disease models

• Translational pathway:

  • Validate findings in human post-mortem tissue

  • Correlate MFSD14B alterations with disease severity

  • Identify potential biomarker applications

Technical innovations:

• Advanced imaging applications:

  • Implement expansion microscopy for nanoscale resolution of MFSD14B in ER subdomains

  • Apply volumetric tissue clearing techniques for whole-brain MFSD14B mapping

  • Develop live-cell reporters for dynamic MFSD14B trafficking studies

• Multi-omics integration:

  • Combine MFSD14B antibody-based proteomics with transcriptomics and metabolomics

  • Apply spatial transcriptomics alongside immunohistochemistry

  • Integrate findings with systems biology approaches to understand pathway interactions

What novel techniques might enhance MFSD14B transport function characterization beyond traditional antibody applications?

While antibodies remain crucial for MFSD14B research, complementary cutting-edge approaches can help elucidate its transport function:

Advanced protein engineering approaches:

• CRISPR-based tagging strategies:

  • Endogenous tagging of MFSD14B for live-cell imaging

  • Split-GFP complementation to visualize protein-protein interactions

  • Proximity labeling (BioID/APEX) for identifying interaction partners in the ER

• Substrate identification methods:

  • Development of transport-dependent sensors

  • Targeted metabolomics comparing MFSD14B-expressing vs. knockout cells

  • Application of click chemistry to identify transported molecules

Structural biology integration:

• Cryo-EM application:

  • Structural determination of MFSD14B in different conformational states

  • Visualization of substrate binding sites

  • Comparison with related MFS transporters of known function

• Computational approaches:

  • Molecular dynamics simulations to predict substrate passage

  • Docking studies with potential substrates

  • Machine learning analysis of transporter-substrate relationships

Functional genomics innovations:

• High-throughput screening designs:

  • CRISPR activation/inhibition screens under metabolic stress

  • Synthetic genetic interaction mapping

  • Parallel reporter assays for regulatory element identification

• Single-cell applications:

  • Single-cell transcriptomics to identify co-regulated gene networks

  • Single-cell proteomics to detect cell-type specific MFSD14B regulation

  • Spatial transcriptomics to map expression patterns at high resolution

Translational research approaches:

• Patient-derived model systems:

  • iPSC-derived neurons from patients with metabolic disorders

  • Organoid models to study MFSD14B in three-dimensional tissue context

  • Humanized mouse models for translational validation

• Multi-modal phenotyping:

  • Correlation of MFSD14B expression with electrophysiological properties

  • Metabolic flux analysis in models with altered MFSD14B expression

  • Behavioral phenotyping of MFSD14B mutant models

Table 6: Emerging Technologies for MFSD14B Functional Characterization