RUFY2 Antibody

What is RUFY2 and why is it relevant to cell biology research?

RUFY2 (RUN and FYVE domain containing 2) is a protein coding gene that belongs to the RUFY family of adaptor proteins. These proteins are characterized by an N-terminal RUN domain, which interacts with small GTP-binding proteins, and a C-terminal FYVE domain involved in the recognition of phosphatidylinositol 3-phosphate . RUFY family proteins play critical roles in regulating endosomal trafficking and cell migration, making them important subjects for research on intracellular transport mechanisms . Understanding RUFY2's function provides insights into fundamental cellular processes that may be dysregulated in various pathological conditions.

What are the key characteristics of RUFY2 protein that affect antibody selection?

When selecting a RUFY2 antibody, researchers should consider several protein characteristics:

• Molecular weight: RUFY2 has a calculated molecular weight of 46 kDa (403 amino acids), but the observed molecular weight in Western blots is typically 65-70 kDa

• Species homology: RUFY2 antibodies may cross-react with human, mouse, and rat RUFY2

• Domain structure: Contains RUN and FYVE domains, with potential epitopes in these regions

• Isoforms: Different splice variants may affect antibody recognition

• Post-translational modifications: May affect antibody binding and observed molecular weight

This discrepancy between calculated and observed molecular weight suggests post-translational modifications that should be considered when interpreting experimental results.

What applications are RUFY2 antibodies validated for?

Based on manufacturer validation data, RUFY2 antibodies are primarily validated for these applications:

Always verify the specific validation data for your application of interest, as performance can vary significantly between manufacturers and antibody clones .

How should I optimize Western blot protocols for RUFY2 detection?

For optimal Western blot detection of RUFY2:

• Sample preparation: Use tissues with known RUFY2 expression (e.g., PC-3 cells, mouse/rat lung tissue, SH-SY5Y cells)

• Protein loading: Load 20-40 μg of total protein per lane

• Gel percentage: Use 10% SDS-PAGE gels for optimal separation

• Transfer conditions: Standard PVDF membrane transfer protocols are sufficient

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody incubation: Start with manufacturer's recommended dilution (typically 1:1000 for WB) , incubate overnight at 4°C

• Detection method: HRP-conjugated secondary antibodies with ECL detection

• Expected band size: Look for bands at 65-70 kDa rather than the calculated 46 kDa

For optimal results, always include positive controls such as PC-3 cells or mouse lung tissue, which have been validated to express detectable levels of RUFY2 .

What considerations should be made when using RUFY2 antibodies for immunohistochemistry?

For successful immunohistochemistry using RUFY2 antibodies:

• Tissue preparation:

  • Fixed tissues: Use 10% neutral buffered formalin

  • Antigen retrieval: Try both citrate buffer (pH 6.0) and TE buffer (pH 9.0) to determine optimal conditions

• Protocol optimization:

  • Start with a dilution range of 1:20-1:200

  • Increase incubation time for weaker signals

  • Test both ABC and polymer detection systems

• Controls:

  • Positive tissue controls: Human testis and brain tissues have been validated

  • Negative controls: Omit primary antibody

  • Comparison controls: If possible, use tissues with known RUFY2 knockout

• Signal interpretation:

  • Understand the expected cellular and subcellular localization pattern

  • Be aware that fixation can affect epitope accessibility

According to validation data, human brain cancer samples have shown positive staining using RUFY2 antibodies at 1:100 dilution .

How can I validate the specificity of my RUFY2 antibody?

To confirm antibody specificity:

• Molecular weight verification: Confirm band appears at expected 65-70 kDa size in Western blot

• Multiple antibody validation: Use antibodies targeting different epitopes of RUFY2

• Genetic approaches:

  • siRNA knockdown: Compare signal between control and RUFY2-depleted samples

  • CRISPR/Cas9 knockout: The gold standard for specificity validation

  • Overexpression: Verify increased signal with RUFY2 overexpression

• Cross-reactivity testing:

  • Test antibody against recombinant RUFY family proteins (RUFY1, RUFY3, RUFY4)

  • Use tissues from different species to assess cross-species reactivity

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

A comprehensive validation should incorporate multiple approaches to ensure antibody specificity and reliability .

How does RUFY2 relate to other RUFY family proteins in experimental design?

When studying RUFY2, important considerations regarding RUFY family proteins include:

• Domain conservation:

  • All RUFY proteins contain RUN and FYVE domains with structural similarities

  • Potential cross-reactivity of antibodies against conserved domains

• Functional overlap:

  • RUFY3 and RUFY4 function as ARL8 effectors in endolysosome positioning

  • RUFY2 may share overlapping functions with other family members

• Expression patterns:

  • RUFY3 has tissue-specific isoforms (neuronal nRUFY3 and immune cell iRUFY3)

  • Consider tissue specificity when designing experiments

• Experimental considerations:

  • Include controls for other RUFY proteins

  • Consider functional redundancy in knockdown experiments

  • Evaluate co-expression patterns in your experimental system

Current research suggests RUFY2's closest paralog is RUNDC3B, while RUFY1 is an important paralog of RUFY3 . This information should guide experimental design, particularly when studying potential redundant functions.

What cellular compartments would you expect to find RUFY2 in, and how does this affect immunostaining protocols?

Based on RUFY protein family characterization:

• Expected subcellular localization:

  • FYVE domain suggests association with phosphatidylinositol 3-phosphate-rich membranes

  • Likely association with endosomal compartments

  • Potential for both cytosolic and membrane-bound pools

• Immunostaining considerations:

  • Fixation: Use paraformaldehyde (4%) to preserve membrane structures

  • Permeabilization: Gentle detergents (0.1% Triton X-100 or 0.1% saponin)

  • Co-staining markers: Include endosomal markers (EEA1, Rab5, Rab7)

• Cell type variations:

  • Expression levels vary across cell types

  • Subcellular distribution may be cell-type specific

  • Consider cell activation state (e.g., LPS stimulation for immune cells)

• Special considerations:

  • Cytoskeleton preservation: Consider using cytoskeleton stabilization buffers

  • Avoid harsh detergents that may disrupt membrane associations

For optimal visualization, a comparison with the localization patterns of other RUFY family members (like RUFY3, which localizes to perinuclear endolysosomal compartments) may provide useful contextual information .

What are the most promising research directions involving RUFY2 antibodies?

Current research suggests several productive areas for RUFY2 investigation:

• Endolysosomal dynamics:

  • Other RUFY family members regulate endolysosomal positioning

  • RUFY2 may play similar roles in specific cellular contexts

• Cell-type specific functions:

  • Expression in neuronal, immune, and epithelial cells

  • Potential role in specialized cell functions

• Disease associations:

  • RUFY2 has been associated with Dyskeratosis Congenita, Autosomal Recessive 2

  • Exploring role in cancer cells (PC-3, SH-SY5Y, U-251)

• Interaction networks:

  • Potential interaction with small GTPases via RUN domain

  • Phosphoinositide binding via FYVE domain

  • Investigation of binding partners using co-immunoprecipitation with RUFY2 antibodies

• Post-translational modifications:

  • Study phosphorylation, ubiquitination patterns

  • Investigate regulation mechanisms

The discrepancy between calculated (46 kDa) and observed (65-70 kDa) molecular weights suggests unexplored post-translational modifications that warrant further investigation .

Why might I observe variations in RUFY2 banding patterns in Western blots?

Multiple factors can contribute to variable RUFY2 banding patterns:

• Post-translational modifications:

  • Phosphorylation, glycosylation, or ubiquitination may alter molecular weight

  • RUFY2's observed weight (65-70 kDa) is significantly higher than calculated (46 kDa)

• Alternative splicing:

  • Multiple splice variants have been reported

  • Different antibodies may recognize different isoforms

• Sample preparation issues:

  • Incomplete denaturation can affect migration

  • Proteolytic degradation may produce fragments

  • Buffer conditions affect protein stability

• Technical considerations:

  • Gel percentage affects resolution

  • Running time and voltage impact band separation

  • Transfer efficiency varies by protein size

• Antibody specificity:

  • Different epitopes may be differentially accessible

  • Cross-reactivity with related proteins (other RUFY family members)

When troubleshooting, systematically optimize sample preparation, electrophoresis conditions, and test multiple antibodies targeting different epitopes of RUFY2.

How can I differentiate between RUFY2 and other RUFY family members in my experiments?

To distinguish RUFY2 from other RUFY family proteins:

• Antibody selection strategies:

  • Choose antibodies raised against non-conserved regions

  • Validate specificity using recombinant proteins

  • Perform peptide competition assays

• Molecular weight differences:

  • RUFY2: Observed at 65-70 kDa

  • RUFY1: ~80 kDa

  • RUFY3: Two isoforms (nRUFY3: 53 kDa, iRUFY3: 74.8 kDa)

  • RUFY4: ~70 kDa

• Expression pattern analysis:

  • RUFY3 isoforms show tissue-specific expression (nRUFY3 in brain, iRUFY3 in immune cells)

  • Compare expression patterns to published data

• Genetic approaches:

  • Use specific siRNAs to confirm band identity

  • Create knockout controls for each family member

  • Use overexpression constructs as positive controls

• IP-MS confirmation:

  • Immunoprecipitate protein and confirm identity by mass spectrometry

Understanding the domain structure and unique regions of each RUFY family member will help design experiments that can reliably distinguish between these related proteins .

How should I address discrepancies between results obtained with different RUFY2 antibodies?

When facing inconsistent results with different RUFY2 antibodies:

• Systematic validation approach:

  • Compare epitope locations of each antibody

  • Evaluate species reactivity differences

  • Check validation data for each application

• Technical considerations:

  • Optimize protocols for each antibody individually

  • Test different fixation/permeabilization methods for IHC/ICC

  • Adjust blocking conditions to reduce background

• Interpretation framework:

  • Antibodies recognizing different epitopes may reveal different aspects of protein biology

  • Some epitopes may be masked by protein interactions or conformations

  • Post-translational modifications may affect epitope accessibility

• Confirmation strategies:

  • Use genetic approaches (knockdown/knockout)

  • Combine antibody-based with non-antibody methods (MS, RNA analysis)

  • Compare with published literature on RUFY2 and related proteins

• Reporting recommendations:

  • Document all antibody details (catalog numbers, lots, dilutions)

  • Report optimization procedures

  • Acknowledge limitations in result interpretation

Remember that different antibodies may recognize different conformations, isoforms, or post-translationally modified forms of RUFY2, which could explain seemingly contradictory results .

How can I use RUFY2 antibodies to investigate potential interactions with ARL8, similar to other RUFY family members?

To investigate RUFY2-ARL8 interactions, similar to those demonstrated for RUFY3/4 :

• Co-immunoprecipitation approach:

  • Use RUFY2 antibodies to pull down protein complexes

  • Probe for ARL8 in immunoprecipitates

  • Include proper controls (IgG control, reverse IP)

• Proximity ligation assay (PLA):

  • Use RUFY2 and ARL8 antibodies from different species

  • Quantify PLA spots in different cellular conditions

  • Compare with known RUFY3-ARL8 interaction as positive control

• Co-localization studies:

  • Perform double immunofluorescence for RUFY2 and ARL8

  • Use high-resolution microscopy (confocal, STED)

  • Quantify co-localization using appropriate statistical methods

• Functional studies:

  • Test if RUFY2 depletion affects ARL8-dependent processes

  • Compare with RUFY3/4 knockout phenotypes

  • Investigate endolysosomal positioning similar to RUFY3 studies

• Biochemical validation:

  • Express recombinant proteins for in vitro binding assays

  • Use purified components to confirm direct interaction

  • Map interaction domains using truncation mutants

Based on findings with RUFY3, include experimental conditions that might enhance interaction detection, such as LPS activation or nutrient starvation, which increased RUFY3-ARL8b interaction in prior studies .

What controls are essential when investigating RUFY2 expression in different tissue and cell types?

When analyzing RUFY2 expression across samples:

• Essential positive controls:

  • Validated RUFY2-expressing samples: PC-3 cells, mouse lung tissue, SH-SY5Y cells

  • Recombinant RUFY2 protein (for antibody validation)

• Negative controls:

  • RUFY2 knockdown/knockout samples

  • Secondary antibody-only controls

  • Isotype controls

• Normalization controls:

  • Loading controls (β-actin, GAPDH) for Western blot

  • Housekeeping gene expression for qPCR

  • Internal tissue controls for IHC

• Cross-validation approaches:

  • Confirm protein expression with multiple antibodies

  • Correlate protein with mRNA expression

  • Compare with publicly available expression databases

• Method-specific controls:

  • For IHC: Include positive tissue controls (human testis, brain)

  • For WB: Include molecular weight markers

  • For qPCR: Include no-RT controls

The importance of proper controls is highlighted by the tissue-specific expression patterns observed in RUFY family proteins, such as the neuronal-specific nRUFY3 and immune cell-specific iRUFY3 isoforms .

How should I design experiments to investigate RUFY2's role in endosomal trafficking based on knowledge of other RUFY proteins?

Based on known functions of other RUFY family members:

• Subcellular localization studies:

  • Co-stain with markers for different endosomal compartments

  • Track changes upon cellular activation (e.g., LPS stimulation)

  • Compare with RUFY3/4 localization patterns

• Functional perturbation approaches:

  • siRNA knockdown or CRISPR/Cas9 knockout of RUFY2

  • Overexpression of wild-type and domain mutants

  • Assess effects on endolysosomal positioning and dynamics

• Cargo trafficking assays:

  • Analyze endocytic uptake and degradation of model cargoes

  • Track recycling pathways (e.g., transferrin recycling)

  • Monitor antigen presentation if working with immune cells

• Live cell imaging:

  • Express fluorescently tagged RUFY2

  • Monitor co-migration with labeled endosomes

  • Quantify movement parameters (velocity, directionality)

• Interaction studies:

  • Investigate binding to small GTPases (via RUN domain)

  • Test phosphoinositide binding (via FYVE domain)

  • Examine cytoskeletal interactions

Consider the findings for RUFY3, which showed recruitment to LAMP1+/ARL8b+ endolysosomes upon LPS activation or nutrient starvation, and affected endolysosomal clustering in the pericentriolar area . Similar mechanisms might apply to RUFY2, possibly in different cell types or conditions.