RANBP9 Antibody

What are the validated applications for RANBP9 antibodies?

RANBP9 antibodies have been validated for multiple experimental applications with specific methodological considerations for each:

It is recommended to titrate antibodies in each testing system to obtain optimal results as sensitivity can be sample-dependent .

What tissues and cell types have shown positive RANBP9 detection?

Researchers should consider the following validated sources when designing experiments:

Positive Western blot detection:

• Mouse brain tissue

• Mouse testis tissue

• HeLa cells

Positive Immunohistochemistry detection:

• Human testis tissue

• Human skin tissue

• Human cervical cancer tissue

• Mouse testis tissue

• Rat testis tissue

Positive Immunofluorescence detection:

• HeLa cells

What is the molecular weight and structure of RANBP9?

When analyzing RANBP9 detection, researchers should note:

• Calculated molecular weight: 729 amino acids, 78 kDa

• Observed molecular weight in SDS-PAGE: 80-90 kDa

• Contains multiple conserved domains (SPRY, LiSH, CTLH and CRA) that serve as scaffolding modules mediating protein-protein interactions

These properties are important for interpreting band patterns in Western blots and understanding potential cross-reactivity.

How should RANBP9 antibodies be stored for optimal performance?

For maintaining antibody integrity:

• Store at -20°C

• Antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Most formulations remain stable for one year after shipment

• Aliquoting is generally unnecessary for -20°C storage

• Some preparations contain 0.1% BSA as stabilizer

How can I validate RANBP9 antibody specificity for protein-protein interaction studies?

When investigating RANBP9 interactions with its binding partners:

• Immunoprecipitation validation approach:

  • Use RanBP9 antibody for IP followed by Western blot detection of suspected interaction partners

  • Include proper negative controls (IgG pulldown)

  • Verify reciprocal interaction by IP with antibodies against suspected partners

  • Validate using RANBP9 knockdown cells as additional controls

• Documented interaction partners to consider as positive controls:

  • Splicing factors (SF3B3, HNRNPM)

  • Poly(A) binding proteins (PABPC1 and PABPC2)

  • ATM (activated following DNA damage)

  • APP, LRP, and β-integrins

Woo et al. demonstrated that RanBP9 forms complexes with APP/BACE1/LRP, validating these interactions through co-immunoprecipitation assays .

What considerations are important when using RANBP9 antibodies in Alzheimer's disease research?

When investigating RANBP9's role in amyloidogenic processing:

• Detection of proteolytic fragments:

  • RanBP9-N60, a 60-kDa proteolytically derived species, binds APP, LRP, and BACE-1 more strongly than full-length RANBP9

  • This fragment is increased 6-fold in AD patient brains

  • Use gel systems capable of resolving proteins in 60-90 kDa range

• Experimental readouts to consider:

  • Analysis of sAPPα levels

  • Aβ generation

  • APP surface levels and endocytosis

  • Synaptic protein markers (PSD-95)

• Cellular localization considerations:

  • Assess nuclear vs. cytoplasmic distribution of RANBP9

  • Evaluate translocation following experimental treatments

What methodology can be used to study RANBP9 phosphorylation in DNA damage response?

For studying RANBP9 in DNA damage response contexts:

• Experimental design for phosphorylation studies:

  • Treat cells with IR (ionizing radiation) to induce DNA damage

  • Use phospho-specific antibodies against S181 and S603 residues of RANBP9

  • Include ATM inhibitors (e.g., KU-55933) as negative controls

  • Monitor co-immunoprecipitation with active ATM

• Downstream markers to assess:

  • ATM phosphorylation (S1981)

  • Chk2 phosphorylation (T68)

  • p53 phosphorylation (S15)

  • γH2AX formation

  • DNA repair efficiency using Comet assay

Research by Palmieri et al. showed that "active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603)" and that "RanBP9 co-immunoprecipitated with active ATM, but not when ATM kinase activity was inhibited" .

What is the optimal protocol for RNA-immunoprecipitation (RIP) to identify RANBP9-bound transcripts?

For researchers investigating RANBP9's role in RNA processing:

• Sample preparation:

  • Homogenize tissue in TNIP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.5% Triton X-100, 0.5% NP-40, 1 mM DTT)

  • Include RNase inhibitors (RNaseOUT 100 U/ml, VRC 400 μM)

  • Add protease inhibitors

  • Use sonication to promote nuclear lysis

• Immunoprecipitation:

  • Use 5 μg validated RANBP9 antibody per reaction

  • Pre-clear lysate with protein G beads

  • Include IgG control immunoprecipitations

  • Incubate antibody-coated beads with nuclear extracts overnight at 4°C

• RNA extraction and analysis:

  • Extract protein-bound RNAs using specialized RNA purification kits

  • Perform qPCR validation of selected targets

  • Consider RNA-Seq for genome-wide identification

Research by Bao et al. identified 2,379 transcripts significantly enriched in RANBP9 immunoprecipitants, many involved in protein/RNA transport and spermatogenesis .

How can I investigate RANBP9's role in alternative splicing?

For studying RANBP9's function in splicing regulation:

• Experimental approach:

  • Generate RANBP9 knockdown or knockout models

  • Perform RNA-Seq analysis to identify changes in splice isoforms

  • Validate altered splicing events using RT-PCR and isoform-specific primers

  • Conduct RIP-Seq to identify directly bound transcripts

• Controls and validation:

  • Confirm physical interaction between RANBP9 and splicing factors (SF3B3, HNRNPM)

  • Verify that expression levels of splicing factors are not affected by RANBP9 manipulation

  • Include rescue experiments with wild-type RANBP9

• Key observations to monitor:

  • "RANBP9 was associated with multiple key splicing factors and directly targeted >2,300 mRNAs in spermatocytes and round spermatids"

  • "Many of the RANBP9 target and non-target mRNAs either displayed aberrant splicing patterns or were dysregulated in the absence of Ranbp9"

How can I study RANBP9's effect on cell surface protein trafficking?

When investigating RANBP9's role in endocytosis:

• Cell surface biotinylation protocol:

  • Culture cells expressing RANBP9 or controls

  • Biotinylate cell surface proteins on ice using Sulfo-NHS-SS-Biotin

  • Immunoprecipitate with anti-biotin antibodies

  • Detect surface levels of proteins of interest (β1-integrin, LRP, APP)

• Endocytosis assay methodology:

  • Surface biotinylate cells with cleavable biotin derivative

  • Allow internalization by returning cells to 37°C for defined time periods

  • Remove non-internalized surface biotin

  • Quantify internalized biotinylated proteins

• Key findings to validate results:

  • "RanBP9 markedly reduced cell surface levels of both LRP and β1-integrin without altering their total cellular content"

  • "RanBP9 accelerated LRP endocytosis by ~50% and β1-integrin endocytosis by ~3-fold"

What methods can be used to visualize RANBP9 localization following stimuli?

For studying dynamic changes in RANBP9 localization:

• Immunofluorescence approach:

  • Treat cells with appropriate stimuli (e.g., IR for DNA damage)

  • Fix cells at different timepoints

  • Perform immunofluorescence using validated RANBP9 antibodies

  • Counterstain with markers for cellular compartments

  • Use confocal microscopy for high-resolution imaging

• Biochemical fractionation alternative:

  • Separate nuclear and cytoplasmic fractions

  • Perform Western blot analysis for RANBP9

  • Include appropriate markers for fraction purity (e.g., Lamin B for nuclear fraction)

• Observation in DNA damage response:

  • "In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition"

How can epitope tagging enhance RANBP9 detection in research models?

For researchers considering genetic modification approaches:

• Benefits of tagged RANBP9 models:

  • More reliable detection in both IHC and WB applications

  • Enhanced immunoprecipitation efficiency for protein-protein interaction studies

  • Overcome limitations of existing antibodies

• Validated tagging approach:

  • C-terminal double (V5-HA) epitope tag fusion to endogenous RANBP9

  • Tag does not interfere with essential functions of RANBP9

  • Tagged mice are viable, fertile and phenotypically normal

• Applications and advantages:

  • "The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB"

  • "Tagged protein pulls down known interactors of wild type RanBP9"

  • Enables discovery of novel interactions (e.g., with Nucleolin)

What considerations are important when using RANBP9 antibodies in cell adhesion studies?

For adhesion and focal adhesion research:

• Experimental design:

  • Plate cells on fibronectin-coated surfaces

  • Quantify cell attachment using colorimetric assays

  • Categorize cell spreading by morphological criteria

  • Include RANBP9 overexpression and knockdown conditions

• Key focal adhesion signaling markers to examine:

  • Pyk2/paxillin signaling

  • Talin/vinculin localization in focal adhesion complexes

• Expected outcomes to validate findings:

  • "RanBP9 overexpression dramatically disrupts integrin-dependent cell attachment and spreading"

  • "RanBP9 knockdown robustly promotes cell attachment, spreading, and focal adhesion signaling and assembly"