FAM107B Antibody

What is FAM107B and what is its significance in research?

FAM107B (family with sequence similarity 107, member B) is a protein with significant research interest, particularly in cancer biology. It has a calculated molecular weight of 16 kDa (131 amino acids), though it is often observed at 38-40 kDa in experimental conditions, suggesting post-translational modifications . Research indicates FAM107B expression is decreased in stomach cancer and various other cancer types, with forced expression diminishing proliferation in response to growth factors, suggesting tumor-suppressive properties . The protein's GenBank accession number is BC004872, and its UniProt ID is Q9H098 . Understanding FAM107B is particularly valuable for researchers investigating cancer proliferation mechanisms and potential therapeutic targets.

How do I select the appropriate FAM107B antibody for my specific research needs?

Selection should be guided by your experimental application and target species. Most commercial FAM107B antibodies show validated reactivity with human and mouse samples . Consider these technical factors:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IP, IF, IHC, ELISA)

• Epitope location: Different antibodies target various epitopes - some target regions 113-131 or 253-306 of human FAM107B

• Antibody class: Determine whether polyclonal (broader epitope recognition) or monoclonal (higher specificity) better suits your needs

• Validation method: Prioritize antibodies validated through multiple methods (siRNA knockdown, independent antibodies, or GFP-tagged verification)

For critical experiments, consider parallel testing of multiple antibodies targeting different protein regions to ensure result consistency.

What explains the discrepancy between calculated (16 kDa) and observed (38-40 kDa) molecular weight of FAM107B?

This significant discrepancy between the calculated 16 kDa molecular weight and the observed 38-40 kDa in experimental conditions warrants methodological consideration:

• Post-translational modifications: FAM107B likely undergoes extensive modifications such as phosphorylation, glycosylation, or ubiquitination

• Protein dimerization: The higher observed weight could result from stable protein dimers forming under experimental conditions

• Experimental factors: SDS-PAGE migration anomalies can occur with hydrophobic or acidic proteins

• Splice variants: Alternative splicing may produce larger isoforms than predicted from reference sequences

To investigate this discrepancy, researchers should:

• Use phosphatase treatment before Western blotting to identify phosphorylation contributions

• Employ reducing versus non-reducing conditions to evaluate multimer formation

• Compare migration patterns across different tissue types to identify tissue-specific modifications

• Verify identity through mass spectrometry analysis of the observed band

What are the optimal conditions for Western blot detection of FAM107B?

For optimal Western blot detection of FAM107B, the following protocol is recommended based on validated approaches:

• Sample preparation:

  • Mouse pancreas tissue has shown reliable positive detection

  • Cell lines such as RT4 (urinary bladder cancer) and U-251 MG (brain glioma) also show detectable expression

• Antibody dilution and incubation:

  • Use antibody at 1:500-1:1000 dilution for Western blot applications

  • Optimal primary antibody incubation is typically overnight at 4°C

• Detection specificity:

  • Expected band at 38-40 kDa (observed molecular weight)

  • Calculated molecular weight is 16 kDa, but this discrepancy is consistent across literature

• Controls:

  • Include positive control tissues (mouse pancreas)

  • Consider using CRISPR knockout samples as negative controls where available

• Optimization note:

  • "It is recommended that this reagent should be titrated in each testing system to obtain optimal results"

How should I design immunofluorescence experiments using FAM107B antibodies?

When designing immunofluorescence experiments with FAM107B antibodies, follow these methodological guidelines:

• Cell preparation:

  • PFA fixation with Triton X-100 permeabilization has been validated for U-251 MG cells (human brain glioma cell line)

  • 2 μg/ml antibody concentration has shown successful detection in IF applications

• Controls and validation:

  • Include known positive cell lines (e.g., U-251 MG)

  • Consider parallel validation using independent antibodies targeting different epitopes

  • For definitive validation, incorporate siRNA knockdown controls or GFP-tagged FAM107B expression

• Co-localization studies:

  • When investigating subcellular localization, pair with established organelle markers

  • Consider dual staining with S100A4 to investigate their reported regulatory relationship

• Imaging parameters:

  • Capture multiple fields to account for heterogeneous expression

  • Use appropriate filter sets to minimize autofluorescence interference

  • Document exposure settings for reproducibility

What methodological approaches work best for FAM107B detection in clinical samples?

For clinical sample analysis of FAM107B, implement these specialized methodological approaches:

• Sample preparation:

  • For FFPE tissue sections, antigen retrieval is crucial as "method used to restore/retrieve the epitope (antibody binding region) of the target protein, cross-linked, and thus masked, during [fixation]"

  • Optimize tissue-specific protocols as FAM107B detection can be sample-dependent

• Validation approach:

  • Use parallel detection methods (IHC and WB from the same specimen when possible)

  • Compare normal vs. cancer tissue to verify reported downregulation in cancer

  • Consider RNA-level validation (qRT-PCR) alongside protein detection

• Interpretation guidelines:

  • Document subcellular localization patterns

  • Quantify expression levels using appropriate scoring systems

  • Compare expression patterns with established markers of disease progression

• Technical considerations:

  • Store antibodies according to manufacturer specifications (-20°C or -80°C depending on formulation)

  • Aliquot antibodies to avoid freeze-thaw cycles, though some formulations note "Aliquoting is unnecessary for -20°C storage"

How can I investigate the functional relationship between FAM107B and S100A4 in cancer biology?

To investigate the FAM107B-S100A4 relationship in cancer, design experiments based on published findings showing that "FAM107B was downregulated by S100A4" and "FAM107B at least partly mediates the effect of S100A4 on the proliferation and migration of MGC803 cells" :

• Expression correlation analysis:

  • Perform qRT-PCR to quantify both FAM107B and S100A4 expression across cancer cell lines

  • Analyze publicly available cancer genomics datasets to examine correlation patterns

• Regulatory mechanism investigation:

  • Use S100A4 siRNA knockdown to confirm upregulation of FAM107B expression

  • Employ chromatin immunoprecipitation to investigate if S100A4 directly or indirectly regulates FAM107B transcription

  • Analyze promoter activity using luciferase reporter assays with FAM107B promoter constructs

• Functional rescue experiments:

  • Replicate the key finding that "FAM107B-siRNA transfection reversed the reduced proliferation and migration abilities induced by S100A4 inhibition"

  • Extend with FAM107B overexpression in S100A4-expressing cells to test antagonistic effects

• Signaling pathway analysis:

  • Investigate downstream effectors common to both proteins

  • Use pharmacological inhibitors of candidate pathways to identify critical nodes

What CRISPR/Cas9 strategies are available for studying FAM107B function?

Multiple CRISPR/Cas9 tools are available for comprehensive genetic manipulation of FAM107B. Design your genetic studies using these approaches:

• Knockout strategies:

  • CRISPR/Cas9 knockout plasmids are available for both human (sc-410178) and mouse (sc-426075) FAM107B

  • Double Nickase plasmid systems offer reduced off-target effects for both species

  • HDR plasmids enable precise genetic modifications with puromycin selection

• Gene activation approaches:

  • CRISPR activation plasmids for transcriptional upregulation are available for both human and mouse FAM107B

  • Lentiviral activation particles provide efficient delivery for difficult-to-transfect cells

  • Activation systems include multiple selection markers (Puromycin, Blasticidin, Hygromycin)

• Experimental design considerations:

  • Include appropriate controls (non-targeting gRNAs)

  • Validate edits through sequencing and expression analysis

  • For cancer studies, consider parallel manipulation of S100A4 given their functional relationship

• Phenotypic analysis framework:

  • Assess effects on cell proliferation, as FAM107B has demonstrated anti-proliferative effects

  • Measure migration capacity through transwell or wound healing assays

  • Evaluate expression of downstream genes potentially regulated by FAM107B

How should I approach antibody validation to ensure FAM107B detection specificity?

Comprehensive validation is essential for confident interpretation of FAM107B antibody results. Implement this multi-tiered validation strategy:

• Genetic validation:

  • Use siRNA knockdown to confirm signal reduction

  • Employ CRISPR knockout cell lines as gold-standard negative controls

  • Overexpression systems can verify antibody detection at expected molecular weight

• Cross-validation with independent antibodies:

  • Use antibodies targeting different epitopes (e.g., those targeting 113-131 vs. 253-306 regions)

  • Compare staining patterns across multiple validated antibodies

  • Assess correlation between antibodies with different host species or clonality

• Application-specific validation:

  • For IF: Confirm co-localization with GFP-tagged FAM107B

  • For WB: Verify expected molecular weight (38-40 kDa observed vs. 16 kDa calculated)

  • For IHC: Compare with RNA expression patterns in matched tissues

• Documentation standards:

  • Record antibody catalog numbers, lot numbers, and dilutions

  • Document validation method results with appropriate controls

  • Consider using the antibody validation reporting guidelines from academic journals

What are common challenges in detecting FAM107B and how can they be addressed?

Researchers frequently encounter these challenges when working with FAM107B antibodies:

• Molecular weight discrepancy:

  • Expected observation at 38-40 kDa despite 16 kDa calculated weight

  • Solution: Include positive control samples with known FAM107B expression; consider denaturing conditions that may affect migration

• Tissue-specific detection issues:

  • "Sample-dependent" reactivity noted in technical documentation

  • Solution: Optimize protein extraction protocols for specific tissue types; pancreatic tissue shows reliable detection

• Antibody specificity concerns:

  • Solution: Validate using independent antibodies targeting different epitopes; consider CRISPR knockout controls

• Storage and stability issues:

  • Different storage recommendations exist (-20°C vs. -80°C)

  • Solution: Follow manufacturer-specific guidelines; for 20527-1-AP, "Stable for one year after shipment. Aliquoting is unnecessary for -20°C storage"

• Conflicting expression data:

  • Solution: Verify expression at both protein and mRNA levels (qRT-PCR) as demonstrated in FAM107B/S100A4 studies

How should I interpret changes in FAM107B expression in cancer studies?

When interpreting FAM107B expression changes in cancer research, consider these analytical frameworks:

• Expression pattern interpretation:

  • Decreased expression has been observed in "stomach cancer and many other kinds of cancer"

  • Consider both magnitude and consistency of expression changes

  • Compare against established cancer markers for contextual interpretation

• Functional significance assessment:

  • "Forced expression of FAM107B in HeLa cells diminished proliferation in response to growth factors"

  • "FAM107B inhibition by siRNA led to significantly increased proliferation and migrating abilities of MGC803 cells"

  • These findings suggest potential tumor suppressor activity

• Regulatory relationship analysis:

  • "FAM107B was an upregulated one" after S100A4 inhibition

  • "FAM107B was downregulated by S100A4"

  • Consider this regulatory relationship when interpreting cancer pathway data

• Clinicopathological correlation:

  • Correlate expression patterns with patient outcomes where available

  • Consider multivariate analysis to determine independent prognostic value

  • Evaluate expression changes in context of tumor grade, stage, and molecular subtype

What experimental controls are critical when studying FAM107B expression and function?

Implement these essential controls for rigorous FAM107B research:

• Expression analysis controls:

  • Positive tissue controls: Mouse pancreas tissue shows reliable detection

  • Positive cell line controls: RT4 cells (urinary bladder cancer) and U-251 MG cells (brain glioma)

  • Loading controls: Traditional housekeeping proteins for normalization

  • Negative controls: Secondary antibody-only controls; ideally CRISPR knockout samples

• Functional study controls:

  • For siRNA experiments: Non-targeting siRNA control

  • For overexpression: Empty vector control

  • For CRISPR: Non-targeting guide RNA control

• Regulatory relationship controls:

  • When studying S100A4-FAM107B relationship:

    • S100A4 inhibition alone

    • FAM107B inhibition alone

    • Combined manipulation as demonstrated in rescue experiments

• Methodology-specific controls:

  • For WB: Molecular weight markers to verify 38-40 kDa observed band

  • For IF: Counterstaining with DAPI for nuclear localization reference

  • For IHC: Adjacent normal tissue as internal reference

How might FAM107B function as a downstream effector in cellular signaling pathways?

Research indicates FAM107B functions as a critical downstream effector in cellular signaling:

• S100A4 pathway involvement:

  • "As a downstream effector, FAM107B at least partly mediates the effect of S100A4 on the proliferation and migration of MGC803 cells"

  • This suggests FAM107B acts as a pathway node translating upstream signals into phenotypic effects

• Growth factor response:

  • "Forced expression of FAM107B in HeLa cells diminished proliferation in response to growth factors"

  • This implies potential involvement in attenuating growth factor signaling cascades

• Research approaches to explore:

  • Phosphoproteomic analysis to identify FAM107B phosphorylation states under various stimuli

  • Protein-protein interaction studies to map FAM107B's signaling partners

  • Transcriptome analysis following FAM107B modulation to identify affected pathways

• Methodological considerations:

  • Use inducible expression systems to control timing of FAM107B expression

  • Employ domain mutation studies to identify functional regions

  • Consider subcellular fractionation to determine compartment-specific interactions

What techniques can help resolve the molecular weight discrepancy of FAM107B?

To investigate the substantial difference between calculated (16 kDa) and observed (38-40 kDa) molecular weight of FAM107B , apply these specialized techniques:

• Post-translational modification analysis:

  • Phosphatase treatment: Compare migration before and after treatment

  • Glycosidase digestion: Assess contribution of glycosylation

  • Mass spectrometry: Identify specific modifications and their locations

• Protein structure evaluation:

  • Native vs. reducing PAGE: Assess impact of disulfide bonding

  • Crosslinking studies: Determine if stable dimers occur

  • Analytical ultracentrifugation: Characterize molecular size in solution

• Transcriptomic verification:

  • RNA-seq analysis to detect potential alternative splicing

  • 5' and 3' RACE to identify potentially unannotated exons

  • Cloning and expression of identified splice variants

• Comparative analysis:

  • Cross-species comparison of migration patterns

  • Cross-tissue analysis to identify tissue-specific forms

  • Comparison across developmental stages for temporal variation