FUS Antibody

What is FUS protein and why are FUS antibodies important in neurodegenerative disease research?

FUS (Fused in Sarcoma, also known as ALS6, ETM4, FUS1, HNRNPP2, RNA-binding protein FUS) is a 53.4 kDa RNA-binding protein that plays essential roles in various cellular processes. FUS was originally discovered as a fusion protein caused by chromosomal translocation in cancer, but has gained significant attention due to its role in neurodegenerative diseases .

FUS mutations, particularly in the C-terminal domain where the nuclear localization signal (NLS) is located, cause redistribution of FUS from the nucleus to the cytoplasm. In neurons, this leads to the formation of neurotoxic aggregates that contribute to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) . The C-terminal end of FUS is involved in protein and RNA binding, while the N-terminal motif plays a role in transcriptional activation .

High-quality FUS antibodies are crucial research tools that enable:

• Detection of normal vs. pathological FUS localization

• Study of FUS aggregation mechanisms

• Investigation of post-translational modifications

• Analysis of FUS interactions with other proteins and nucleic acids

How do I select the most appropriate FUS antibody for my specific research application?

Selecting the appropriate FUS antibody requires consideration of several factors:

Epitope location:

• N-terminal antibodies (e.g., targeting regions AA 1-198) are useful for detecting total FUS regardless of C-terminal mutations

• C-terminal antibodies (e.g., targeting regions AA 499-526) are valuable for studying NLS mutations and nuclear-cytoplasmic distribution

Application compatibility:
Based on systematic antibody characterization studies, not all antibodies perform equally across applications :

Clonality:

• Monoclonal antibodies (e.g., clone 10F7, 4H11) offer high specificity and batch consistency

• Polyclonal antibodies may provide stronger signals but with potential for more background

Host species:
Consider secondary antibody compatibility and avoid host interference if co-staining with other antibodies .

What are the most reliable validation methods to ensure FUS antibody specificity?

Reliable FUS antibody validation should incorporate multiple approaches:

Knockout cell line validation:
The gold standard approach involves comparing antibody signals in wildtype vs. FUS knockout cells :

• HeLa WT and FUS KO cell lines are commonly used for validation

• Signals should be present in WT cells and absent in KO cells

• This approach effectively eliminates false positives from antibodies that cross-react with other proteins

Mosaic strategy for immunofluorescence:

• Label WT and KO cells with different fluorescent dyes (e.g., green for WT, far-red for KO)

• Mix and plate cells together at 1:1 ratio on coverslips

• Perform immunofluorescence with the FUS antibody

• Image both cell types in the same field of view to reduce staining biases

• Confirm specific staining in WT cells with absence in KO cells

Western blot validation:

• Run WT and KO cell lysates side by side

• Confirm band at expected molecular weight (~70 kDa) in WT lanes only

• Include Ponceau staining to confirm equal loading and transfer efficiency

Application-specific validation:
For applications like immunoprecipitation:

• Evaluate antibody performance by assessing FUS depletion from extracts

• Analyze FUS detection in starting material, unbound fraction, and immunoprecipitate

How can I optimize Western blot protocols specifically for FUS detection?

Optimizing Western blot protocols for FUS detection requires attention to several key factors:

Lysate preparation:

• Use cell lysis buffers containing protease inhibitors to prevent degradation

• For phosphorylation studies, include phosphatase inhibitors (e.g., calyculin-A)

• Load 30 μg of protein per lane for optimal detection

Electrophoresis conditions:

• Note that FUS often runs at ~70 kDa despite its predicted size of 53.4 kDa

• Use appropriate percentage gels (8-10%) to achieve good resolution in this molecular weight range

Antibody selection and dilution:
Based on validation studies, recommended antibodies and dilutions include:

• NBP2-52874 at 1/1000 or 1/2000

• GTX101810 at 1/3000 (note: titration required as supplier's recommendation may result in weak signal)

• 60160-1-Ig at 1/10000

• 11570-1-AP at 1/4000

• MA3-089 at 1/2000

• ab243880 at 1/1000 or 1/2000

Controls:

• Include positive control (WT cell lysate)

• Include negative control (FUS KO cell lysate if available)

• Use Ponceau staining to confirm equal loading and transfer efficiency

• For phosphorylation studies, include samples treated with phosphatase

Signal detection:

• If studying post-translational modifications, look for band shifts

• For phosphorylation analysis, use phospho-specific antibodies alongside total FUS antibodies

What are the best practices for immunofluorescence with FUS antibodies?

Successful immunofluorescence with FUS antibodies requires careful attention to protocol details:

Cell preparation:

• For validation studies, use the mosaic approach: label WT and KO cells with different fluorescent dyes (e.g., green and far-red), mix at 1:1 ratio, and plate on same coverslip

• For regular experiments, grow cells on glass coverslips for 24 hours in standard conditions (37°C, 5% CO₂)

Fixation and permeabilization:

• Fix cells in 4% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature

• Wash 3 times with PBS

• Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes at room temperature

Blocking:

• Block with PBS containing 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 minutes at room temperature

Antibody incubation:

• Prepare primary antibodies in IF buffer (PBS with 5% BSA and 0.01% Triton X-100)

• Incubate overnight at 4°C

• Wash 3 times for 10 minutes each with IF buffer

• Incubate with appropriate secondary antibodies (e.g., Alexa Fluor 555-conjugated) at 1.0 μg/mL for 1 hour at room temperature

• Include DAPI staining during secondary antibody incubation

• Wash 3 times for 10 minutes each with IF buffer, then once with PBS

Antibody selection and dilution:
Several antibodies perform well in immunofluorescence:

• NBP2-52874 (monoclonal)

• GTX101810 (requires titration)

• ab243880 (recombinant)

• 60160-1-Ig (monoclonal)

Analysis considerations:

• Normal FUS localization is predominantly nuclear

• In disease states or after specific treatments, look for cytoplasmic mislocalization or aggregates

• For DNA damage response studies, monitor both phosphorylation and localization patterns

How can I use FUS antibodies to study its role in DNA damage response?

FUS plays important roles in DNA damage response, and antibodies can be valuable tools to study these functions:

Monitoring phosphorylation:

• FUS becomes multiphosphorylated following DNA damage, particularly in its prion-like domain (PrLD)

• At least 28 putative phosphorylation sites have been identified, with approximately half being DNA-dependent protein kinase (DNA-PK) consensus sites

• Custom phospho-specific antibodies (e.g., targeting Ser-26 and Ser-30) can detect specific phosphorylation events

Experimental design:

• Treatment protocols:

  • Use appropriate DNA-damaging agents (e.g., calicheamicin)

  • Consider using calyculin-A to inhibit phosphatases and preserve phosphorylation states

• Antibody selection:

  • Use phospho-specific antibodies to detect specific modifications

  • Pair with total FUS antibodies to normalize levels

  • Look for band shifts in Western blots that indicate phosphorylation

• Controls:

  • Include untreated controls (phospho-specific antibodies should not recognize these)

  • Consider siRNA knockdown controls to confirm antibody specificity

  • For mechanistic studies, include DNA-PK inhibitors to confirm pathway involvement

• Localization studies:

  • Monitor both phosphorylation state and subcellular localization

  • Note that DNA-PK-dependent multiphosphorylation of FUS's prion-like domain does not necessarily cause cytoplasmic localization

Analysis considerations:

• Phosphorylation may occur in only a subpopulation of cellular FUS following DNA damage

• Different phosphorylation sites may not be modified equally or simultaneously

• Consider the relationship between phosphorylation patterns and FUS function in DNA repair

What is known about FUS antibody cross-reactivity with other proteins and how can I address it?

FUS antibody cross-reactivity can complicate experimental interpretation, particularly since FUS belongs to the FET family of RNA-binding proteins with similar structural features:

Known cross-reactivity issues:

• Some FUS antibodies may cross-react with other FET family members (TAF15, EWSR1) due to sequence homology

• Antibodies targeting highly conserved regions are more prone to cross-reactivity

• Non-specific binding can also occur to proteins with similar epitope structures

Strategies to address cross-reactivity:

• Use KO-validated antibodies:

  • Antibodies validated using FUS knockout cells provide the highest confidence in specificity

  • Look for antibodies that show no signal in FUS KO cells

• Epitope selection:

  • Choose antibodies targeting unique regions of FUS with less homology to other proteins

  • Consider the specific amino acid sequences used as immunogens when selecting antibodies

• Multiple antibody approach:

  • Use multiple antibodies targeting different epitopes of FUS

  • Consistent results across different antibodies increase confidence in findings

• Complementary techniques:

  • Combine antibody-based detection with other techniques (e.g., mass spectrometry)

  • For genetic studies, use mRNA detection methods alongside protein detection

• Blocking peptides:

  • Use specific blocking peptides when available (e.g., AAP40278 for ARP40278_P050 antibody)

  • This can help determine if binding is specific to the target epitope

Documentation and reporting:

• Clearly document which antibody was used (catalog number, lot, clone)

• Report validation methods used to confirm specificity

• Consider the predicted species reactivity when working with non-human models

How do I properly design immunoprecipitation experiments using FUS antibodies?

Effective immunoprecipitation (IP) of FUS requires careful experimental design:

Antibody selection:

• Not all FUS antibodies perform equally in IP applications

• In systematic evaluations, several antibodies showed strong IP performance:

  • NBP2-52874 (monoclonal)

  • ab243880 (recombinant)

  • 4H11 (monoclonal, from Santa Cruz)

Protocol optimization:

• Sample preparation:

  • Use appropriate lysis buffers that maintain protein-protein interactions of interest

  • For nuclear proteins like FUS, ensure effective nuclear lysis

  • Include protease inhibitors to prevent degradation

  • For phosphorylation studies, include phosphatase inhibitors

• Antibody coupling:

  • Pre-couple 1.0 μg of FUS antibody to protein G or protein A Sepharose beads

  • For mouse monoclonal antibodies, protein G is generally preferred

  • For rabbit antibodies, both protein A and G can work well

• IP procedure:

  • Include appropriate controls (e.g., IgG control, input sample)

  • Save fractions for analysis: starting material (SM), unbound fraction (UB), and immunoprecipitate (IP)

  • Load 10% of starting material and unbound fraction for comparison

• Detection:

  • Use Western blotting to detect FUS in all fractions

  • Consider using a different antibody for detection than was used for IP

  • Look for efficient depletion from the unbound fraction and enrichment in the IP fraction

Performance evaluation:
A successful IP should show:

• Significant reduction of FUS in the unbound fraction compared to starting material

• Strong enrichment of FUS in the immunoprecipitate

• Minimal background in negative controls

What are the key considerations for studying FUS mutations and aggregation in disease models?

Studying FUS mutations and aggregation patterns in disease models presents several unique challenges:

Antibody considerations for mutation studies:

• Epitope location:

  • Ensure the antibody epitope is not affected by the mutation of interest

  • For C-terminal NLS mutations common in ALS/FTD, use antibodies targeting N-terminal or middle regions

  • For studying specific mutations, consider whether the mutation might alter antibody binding

• Detection of aggregates:

  • Some antibodies may have differential access to epitopes in aggregated vs. soluble FUS

  • Consider using multiple antibodies targeting different regions

  • Native vs. denatured conditions may affect epitope accessibility

Experimental approaches:

• Cellular models:

  • Immunofluorescence can detect FUS mislocalization from nucleus to cytoplasm

  • Look for cytoplasmic aggregates in neuronal models expressing mutant FUS

  • Monitor co-localization with stress granule markers

• Biochemical analysis:

  • Use differential solubility assays to separate aggregated from soluble FUS

  • Western blot analysis can detect shifts in molecular weight or solubility

  • Immunoprecipitation can identify altered protein interactions

• Controls and comparisons:

  • Always include wild-type FUS controls

  • Compare multiple FUS mutations to identify mutation-specific effects

  • In patient-derived samples, compare to age-matched controls

Analytical considerations:

• FUS normally localizes predominantly to the nucleus

• In disease states, look for:

  • Cytoplasmic mislocalization

  • Formation of stress granule-like structures

  • Insoluble aggregates

  • Post-translational modifications

  • Altered protein-protein interactions

How can I quantitatively assess FUS protein levels and localization in cellular models?

Quantitative assessment of FUS levels and localization requires rigorous methodological approaches:

Protein level quantification:

• Western blot quantification:

  • Use validated antibodies with linear response ranges

  • Include loading controls (e.g., β-actin, GAPDH) for normalization

  • Use standard curves with recombinant protein for absolute quantification

  • Employ image analysis software for densitometry measurements

• Flow cytometry:

  • Several antibodies perform well in flow cytometry: NBP2-52874, NBP2-76415, and AFFN-FUS antibodies

  • Use fluorochrome-conjugated secondary antibodies (e.g., CoraLite® Plus 647)

  • Include secondary-only controls

  • Compare staining intensity between populations (e.g., WT vs. KO, treated vs. untreated)

Localization analysis:

• Immunofluorescence quantification:

  • Perform z-stack imaging to capture the full cellular volume

  • Use nuclear markers (e.g., DAPI) to define nuclear boundaries

  • Calculate nuclear/cytoplasmic ratios of FUS signal intensity

  • Use automated image analysis for unbiased quantification

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions biochemically

  • Perform Western blot analysis on each fraction

  • Include fraction-specific markers to confirm clean separation

  • Calculate nuclear/cytoplasmic distribution ratios

Analytical considerations:

• For localization studies, analyze sufficient cells for statistical power

• Report both mean/median values and distribution patterns

• Consider heterogeneity within cell populations

• For time-course studies, include multiple timepoints to capture dynamics

Statistical analysis:

• Perform appropriate statistical tests based on data distribution

• For comparing multiple conditions, use ANOVA with suitable post-hoc tests

• Report p-values and confidence intervals

• Consider biological vs. technical replicates in experimental design