FUS Antibody Pair

What is FUS protein and why is it significant in research?

FUS (Fused in Sarcoma) is an RNA-binding protein involved in various cellular processes including transcription, RNA processing, and DNA repair mechanisms. It has gained significant research interest due to its association with neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS). FUS contains a prionlike domain (PrLD) that can become multiphosphorylated following DNA damage, making it an important protein to study in both normal cellular functions and disease states . Detection and analysis of FUS requires specific antibodies that can recognize this protein in various experimental contexts.

What applications are FUS antibodies typically used for?

FUS antibodies are employed in multiple laboratory techniques. According to product information, common applications include Western Blot (WB) with recommended dilutions of 1:500-1:2000, Immunohistochemistry (IHC) at 1:20-1:200 dilutions, and Immunofluorescence (IF) at 1:50-1:200 dilutions . Additionally, certain conjugated FUS antibodies (such as HRP-conjugated or Biotin-conjugated variants) are specifically optimized for ELISA applications . These applications allow researchers to detect, quantify, and visualize FUS protein in various experimental systems.

How do I select the appropriate FUS antibody for my experiment?

Selection of FUS antibodies should be based on several factors:

• The specific application (WB, IHC, IF, ELISA)

• The species of your sample (human, mouse, etc.)

• The cellular compartment being studied (nuclear vs. cytoplasmic FUS)

• Whether you need to detect specific post-translational modifications

For human FUS detection, antibodies raised against Homo sapiens FUS are available, such as those raised in rabbit hosts . Different applications require different antibody dilutions, and some may benefit from specific conjugates such as HRP, FITC, or Biotin, depending on your detection system .

What are the recommended dilutions for different FUS antibody applications?

Based on product specifications, the following dilutions are recommended for FUS antibodies:

These ranges provide starting points that should be optimized for your specific experimental conditions and antibody lot .

How can I detect phosphorylated FUS and what significance does it have?

Detection of phosphorylated FUS requires specialized phospho-specific antibodies. Research has demonstrated that FUS can be multiphosphorylated following DNA damage, particularly at sites in its prionlike domain (PrLD). Custom antibodies have been developed to detect specific phosphorylation sites such as Ser-26 and Ser-30 . These phosphorylation events occur in a subpopulation of cellular FUS following DNA-damaging stresses but not necessarily equally or simultaneously.

Methodologically, researchers have confirmed phosphorylation using:

• Mass spectrometry analysis of immunoprecipitated FUS

• Custom antibodies against phosphorylated epitopes

• Immunoblotting techniques comparing phosphorylated vs. non-phosphorylated states

Phosphorylation of FUS's PrLD appears to affect its liquid-liquid phase separation (LLPS) properties and aggregation tendencies, suggesting potential therapeutic implications for inhibiting pathological aggregation in diseases like ALS .

How do FUS antibodies perform in detecting FUS protein interactions?

FUS interacts with numerous proteins involved in RNA metabolism, transcription, and other cellular processes. When studying these interactions, researchers have successfully employed FUS antibodies in immunoprecipitation experiments followed by mass spectrometry or immunoblotting.

In one significant study, mouse anti-FUS antibody (Santa Cruz, sc-47711) was used to immunoprecipitate endogenous FUS from SH-SY5Y cells . This approach successfully co-precipitated FUS interaction partners including DHX9, Matrin-3, ILF2, and hnRNPA1 . The interactions were further validated using GST-FUS pull-down followed by immunoblotting with specific antibodies against these partners.

Interestingly, the study revealed that FUS interactions are differentially affected by RNase treatment:

• Interactions with DHX9, Matrin-3, and DDX3X were enhanced by RNase

• Interactions with Caprin-1 and ILF2 were weakened but not eliminated

• Interaction with hnRNPA1 was completely RNA-dependent

This demonstrates the value of FUS antibodies in distinguishing between direct protein-protein interactions and RNA-mediated associations.

What is the significance of FUS interactions with ALS-related proteins?

FUS antibodies have helped reveal important interactions between FUS and other ALS-associated proteins, notably Matrin-3 and hnRNPA1, suggesting common pathogenic pathways in ALS . These interactions show distinct characteristics:

• FUS-Matrin-3 interaction is enhanced by RNase treatment, suggesting a direct protein-protein interaction

• FUS-hnRNPA1 interaction is entirely RNA-dependent

• Both hnRNPA1 and FUS localize to stress granules under certain conditions

These findings highlight the utility of FUS antibodies in elucidating disease mechanisms and potential therapeutic targets. The analysis of FUS-Matrin-3 and FUS-hnRNPA1 complexes in ALS pathogenesis continues to be an active area of research .

How should I design experiments to study FUS phosphorylation patterns?

To study FUS phosphorylation patterns, researchers have employed several methodological approaches:

• Mass spectrometry analysis: Immunoprecipitate FUS (using anti-FUS antibodies) from cells treated with DNA-damaging agents such as calicheamicin or camptothecin, followed by mass spectrometry to identify phosphorylation sites .

• Phospho-specific antibodies: Develop or obtain antibodies specifically recognizing phosphorylated residues (e.g., phospho-Ser-26 and phospho-Ser-30) for immunoblotting .

• Comparative analysis: Compare phosphorylation patterns following various treatments, such as DNA-damaging agents (calicheamicin, camptothecin), phosphatase inhibitors (calyculin-A), or kinase inhibitors (staurosporine) .

Research has shown that different treatments may affect the total cellular FUS differently, with some causing band shifts visible in standard immunoblotting and others causing more subtle changes detectable only with phospho-specific antibodies .

What controls should I include when using FUS antibodies for immunoprecipitation?

When performing immunoprecipitation with FUS antibodies, several critical controls should be included:

• Negative control antibody: Use an isotype-matched irrelevant antibody (e.g., mouse anti-HA antibody has been used as a negative control for mouse anti-FUS immunoprecipitation) .

• Pre-clearing step: Pre-clear cellular lysates with Protein G resin before immunoprecipitation to reduce non-specific binding .

• Input sample: Always include an aliquot of the starting material to verify the presence of target proteins before immunoprecipitation.

• RNase-treated samples: Include parallel samples with and without RNase treatment to distinguish RNA-dependent from direct protein-protein interactions .

• Validation by immunoblotting: Confirm the identity of co-precipitated proteins using specific antibodies against suspected interaction partners .

These controls help ensure the specificity and reliability of FUS immunoprecipitation results and enable proper interpretation of protein interaction data.

What considerations are important when selecting antibody pairs for FUS detection in sandwich assays?

When selecting antibody pairs for sandwich immunoassays (like sandwich ELISA) targeting FUS protein:

• Epitope compatibility: Choose antibodies recognizing non-overlapping epitopes on FUS to allow simultaneous binding. The FUS protein has distinct domains including an N-terminal prionlike domain (PrLD), RNA recognition motifs, and a C-terminal nuclear localization signal that can be targeted by different antibodies .

• Conjugate selection: For capture-detection pairs, consider conjugated antibodies for detection. Options include HRP-conjugated (CSB-PA02704B0Rb), FITC-conjugated (CSB-PA02704C0Rb), or Biotin-conjugated (CSB-PA02704D0Rb) FUS antibodies .

• Antibody origin: Ideally, use antibodies from different host species to avoid cross-reactivity in secondary detection systems.

• Validation: Validate the antibody pair's performance using recombinant FUS protein and lysates from cells known to express FUS at varying levels.

• Sensitivity and specificity: Test the pair's limits of detection and potential cross-reactivity with related proteins like TDP-43 or other RNA-binding proteins.

The combination of these considerations will help establish a reliable sandwich immunoassay system for specific FUS detection.

How do I address inconsistent results when detecting phosphorylated FUS?

Inconsistent results when detecting phosphorylated FUS may stem from several factors:

• Subpopulation dynamics: Research has shown that phosphorylation occurs in a subpopulation of cellular FUS following DNA damage, and not all sites are necessarily phosphorylated equally or simultaneously . This heterogeneity can lead to variable results.

• Treatment conditions: Different DNA-damaging agents may produce different phosphorylation patterns. For example, calicheamicin, calyculin-A, and staurosporine have been shown to have more extreme effects on total cellular FUS compared to other treatments .

• Antibody sensitivity: Standard FUS antibodies may not detect subtle band shifts caused by phosphorylation. Using phospho-specific antibodies (like those against phospho-Ser-26 or phospho-Ser-30) may be necessary to detect these modifications .

• Timing considerations: The timing of phosphorylation events after DNA damage may vary. Consider performing time-course experiments to capture the dynamic nature of FUS phosphorylation.

• Phosphatase activity: Endogenous phosphatases may rapidly remove phosphate groups from FUS. Consider including phosphatase inhibitors in your sample preparation.

What are the implications of RNA-dependent versus direct protein interactions with FUS?

Understanding whether FUS interactions are RNA-dependent or direct protein-protein interactions has important implications:

• Experimental design: RNase treatment can dramatically alter the interaction profile of FUS. Some interactions (like with DHX9, Matrin-3, and DDX3X) are enhanced by RNase treatment, while others (like with Caprin-1 and ILF2) are weakened but not eliminated, and still others (like with hnRNPA1) are completely abolished .

• Biological interpretation: RNA-dependent interactions suggest functional relationships in RNA metabolism, while direct protein interactions may indicate structural or regulatory relationships independent of RNA.

• Disease mechanisms: The differential nature of FUS interactions with ALS-related proteins like Matrin-3 (enhanced by RNase) and hnRNPA1 (RNA-dependent) may suggest distinct pathogenic mechanisms .

• Therapeutic targeting: Understanding the nature of FUS interactions helps in designing therapeutic approaches. For example, targeting direct protein interactions might require different strategies than disrupting RNA-dependent complexes.

• Subcellular localization: FUS can interact with proteins that primarily localize to different cellular compartments. For instance, predominantly nuclear WT FUS can interact with the almost exclusively cytoplasmic DDX3X or Caprin-1 , highlighting the importance of examining these interactions in cellular contexts.

How should I interpret contradictory results between in vitro and cellular FUS antibody studies?

When facing contradictions between in vitro and cellular studies using FUS antibodies:

• Context-dependent interactions: FUS interactions are highly dynamic and context-dependent. Research has shown that FUS cycles between different molecular complexes and ribonucleoprotein granules depending on cellular conditions, subcellular localization, and stress conditions .

• RNA binding states: FUS displays differential affinities to protein partners in RNA-bound versus RNA-unbound states, suggesting that protein interactions change during RNA binding cycles . In vitro studies may not recapitulate this complexity.

• Post-translational modifications: Modifications like phosphorylation can dramatically alter FUS properties. DNA-PK-dependent multiphosphorylation of FUS's prionlike domain affects its behavior , which may not be captured in simplified in vitro systems.

• Extraction methods: The method used to generate cellular lysates affects which proteins are efficiently extracted. Methods that extract both cytosolic and nuclear proteins (like those used in some studies) enable detection of interactions between proteins that normally reside in different cellular compartments .

• Validation approaches: To resolve contradictions, employ multiple complementary techniques such as immunoprecipitation, GST pull-down, and imaging techniques to determine whether proteins interact in live cells under physiological conditions .

What are emerging applications for FUS antibody pairs in neurodegenerative disease research?

Emerging applications of FUS antibody pairs in neurodegenerative disease research include:

• Biomarker development: Paired antibodies targeting total and phosphorylated FUS could serve as diagnostic or prognostic biomarkers for ALS and frontotemporal dementia. Research has identified multiple phosphorylation sites in the FUS PrLD that may be disease-relevant .

• Monitoring disease progression: Antibody pairs that can distinguish between normal and pathological FUS conformations or modifications could help monitor disease progression and treatment response.

• Therapeutic development: Antibodies targeting specific FUS domains or conformations might have therapeutic potential by preventing pathological aggregation. Research has shown that phosphorylation of the PrLD dramatically reduces liquid-liquid phase separation and formation of solid toxic aggregates .

• Studying FUS-ALS protein connections: Antibody pairs can help elucidate the relationships between FUS and other ALS-related proteins like Matrin-3 and hnRNPA1 , potentially revealing common pathogenic mechanisms.

• Stress granule dynamics: Using antibody pairs to simultaneously track FUS and stress granule markers could provide insights into how FUS contributes to stress granule formation and dysfunction in disease contexts.

How might advances in FUS antibody technology improve our understanding of liquid-liquid phase separation?

Recent research has revealed the importance of FUS in liquid-liquid phase separation (LLPS) processes, with implications for both normal cellular function and disease:

• Phosphorylation-sensitive detection: Antibodies that can specifically detect phosphorylated forms of FUS could help track how post-translational modifications affect LLPS. Research has shown that phosphomimetic substitutions in FUS's PrLD dramatically reduce LLPS and formation of solid toxic aggregates .

• Conformation-specific antibodies: Developing antibodies that recognize specific conformational states of FUS during phase separation could provide real-time visualization of these dynamic processes.

• Multi-epitope detection: Antibody pairs targeting different regions of FUS could reveal how domain-specific interactions contribute to LLPS and transitions to more solid states.

• Quantitative analysis: Advances in quantitative immunoassays using FUS antibody pairs could provide precise measurements of phase-separated versus diffuse FUS under various conditions.

• Therapeutic targeting: Understanding how different regions of FUS contribute to LLPS could guide the development of therapeutic antibodies or other molecules that modulate this process in disease contexts.