RANBP9 Antibody, HRP conjugated

What are the validated applications for RanBP9 antibodies and which dilutions should be used?

RanBP9 antibodies, including HRP-conjugated variants, have been validated for several applications with specific optimal dilution ranges:

Antibody titration is essential for each experimental system as optimal concentrations may vary with sample type, fixation method, and detection system. HRP-conjugated antibodies are particularly useful for direct detection in Western blot applications, eliminating the need for secondary antibodies and potentially reducing background.

Why is it challenging to detect RanBP9 and what strategies can improve detection?

Detecting RanBP9 presents several technical challenges:

• Paralog interference: RanBP9's paralog, RanBP10, shows extensive protein similarity, limiting the specific sequences available for raising antibodies .

• Antibody limitations: "Many commercially available αRANBP9 antibodies fall short in their detection power, reliability, and/or specificity" .

• Signal-to-noise ratio: Even validated antibodies like HPA050007 may show low signal-to-noise ratio in Western blots .

Methodological improvements include:

• Using epitope-tagged versions of RanBP9 (e.g., V5-HA tags)

• Including proper controls (RanBP9 knockout/knockdown samples)

• Optimizing antigen retrieval for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Using multiple antibodies targeting different epitopes to confirm results

• Proper sample preparation with protease and phosphatase inhibitors

The RanBP9-TT mouse model with endogenous RanBP9 fused to V5-HA tags represents a valuable tool that "allows unequivocal detection of RanBP9 both by IHC and WB" .

What controls should be included when working with RanBP9 antibodies?

Rigorous controls are essential when working with RanBP9 antibodies:

For co-immunoprecipitation experiments, researchers have demonstrated successful validation by showing that "RanBP9 is robustly enriched and ranks first for abundance in RanBP9-TT fractions while is absent in the RanBP9 WT MEF IPed extracts" .

How can I design experiments to investigate RanBP9's role in the CTLH complex using RanBP9 antibodies?

The CTLH complex is a multi-subunit E3 ubiquitin-protein ligase complex where RanBP9 serves as a core component. A comprehensive experimental approach includes:

• Co-immunoprecipitation strategy:

  • Immunoprecipitate RanBP9 using validated antibodies

  • Probe for known CTLH complex members (Gid8, Muskelin, Maea, Armc8, Wdr26, Rmnd5A)

  • Validate with reciprocal co-IPs using antibodies against other complex members

• Mass spectrometry verification:

  • IP-MS/MS analysis has identified all CTLH complex members after RanBP9 immunoprecipitation

  • When ranking proteins by abundance, "all 11 members of the CTLH complex are present within the top 20 hits"

• Functional characterization:

  • Analyze effects of RanBP9 depletion on CTLH complex assembly

  • Investigate E3 ligase activity toward known substrates (e.g., transcription factor HBP1)

  • Use domain mutants to map regions required for complex formation

The RanBP9-TurnX mouse model, where "RanBP9 fused to three copies of the HA tag can be turned into RanBP9-V5 tagged upon Cre-mediated recombination," enables cell type-specific biochemical studies of CTLH complex composition and function .

What approaches can reveal RanBP9's role in amyloidogenic processing of APP and Aβ generation?

RanBP9 significantly influences APP processing and Aβ generation through several mechanisms. Experimental strategies include:

• Protein interaction analysis:

  • Co-immunoprecipitate RanBP9 with APP, LRP, and BACE1

  • Domain mapping reveals that "the C37 region of LRP functions to stabilize the interaction between RanBP9 and APP"

  • Determine binding affinities and interaction dynamics

• APP processing assessment:

  • Measure Aβ40 and Aβ42 levels by ELISA in RanBP9-overexpressing/depleted systems

  • Analyze APP cleavage products (sAPP-α, sAPP-β, CTFs) by Western blot

  • RanBP9 overexpression "markedly increased the secretion of sAPP-β and reduced sAPP-α without changing sAPP total"

• Mechanistic investigation:

  • Track APP trafficking using cell surface biotinylation and internalization assays

  • Monitor BACE1 activity using fluorogenic substrates

  • Analyze effects on γ-secretase processing

• In vivo validation:

  • RanBP9 transgenic mice show ">2-fold increase in Aβ40 levels as early as 4 mo of age"

  • Sustained increases in both CHAPS-soluble and formic acid-soluble Aβ fractions

  • Correlate with increased amyloid plaque deposition and decreased synaptic proteins

These approaches collectively demonstrate that "RanBP9 strongly increased BACE1 cleavage of APP and Aβ generation" .

How can I differentiate between RanBP9 and its paralog RanBP10 in experimental settings?

Distinguishing between these highly similar proteins requires strategic approaches:

• Antibody-based methods:

  • Select antibodies raised against divergent regions

  • Validate using knockout controls

  • As demonstrated in one study, antibody specificity was confirmed when "the protein immunoprecipitated from RanBP10 WT/RanBP9-TT MEF cell lysates" disappeared in "RanBP10 knockout/RanBP9-TT double mutant MEFs"

• Genetic approaches:

  • Use specific siRNA/shRNA sequences targeting unique regions

  • Design RT-qPCR primers spanning non-homologous regions

  • Create single and double knockout models via CRISPR-Cas9

• Protein analysis methods:

  • Exploit subtle differences in molecular weight (RanBP9: 80-90 kDa)

  • Use mass spectrometry to identify unique peptides

  • Two-dimensional gel electrophoresis to separate based on charge and size

• Tagged protein strategies:

  • Express epitope-tagged versions for unambiguous detection

  • The RanBP9-TT mouse model with V5-HA tags enables reliable distinction

For comprehensive analysis, combine multiple approaches and include appropriate controls to ensure conclusive identification of each protein.

What methodological approaches can reveal RanBP9's role in DNA damage response?

RanBP9 has been identified as a "novel mediator of cellular DNA damage response" . Strategic experimental approaches include:

• Phosphorylation analysis:

  • ATM phosphorylates RanBP9 at "at least two different residues (S181 and S603) following IR exposure"

  • Generate phospho-specific antibodies against these sites

  • Compare phosphorylation kinetics with other ATM substrates

• Protein interaction studies:

  • "RanBP9 co-immunoprecipitated with active ATM" after IR treatment

  • This interaction was abolished by the ATM inhibitor KU-55933

  • Analyze changes in RanBP9 interactome after DNA damage using IP-MS/MS

• Functional characterization:

  • Create phospho-mimetic (S→D) and phospho-deficient (S→A) mutants

  • Assess effects on DNA repair efficiency and cell survival

  • Compare with known ATM-dependent DNA damage response pathways

• Localization analysis:

  • Track RanBP9 recruitment to DNA damage sites

  • Co-localization with γH2AX, 53BP1, and other damage markers

  • Compare dynamics in ATM-proficient vs. ATM-deficient cells

These approaches will elucidate whether RanBP9 functions as a signaling intermediate, scaffold protein, or direct effector in the DNA damage response pathway.

What explains the multiple bands often observed in RanBP9 Western blots?

Multiple bands in RanBP9 Western blots can result from several biological and technical factors:

• Proteolytic processing:

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

  • This fragment is "increased 6-fold in the brains of patients with AD"

  • May represent functionally relevant species

• Post-translational modifications:

  • Phosphorylation by ATM at S181 and S603

  • Potential ubiquitination as part of the CTLH E3 ligase complex

  • Other modifications affecting electrophoretic mobility

• Isoforms and splice variants:

  • Calculated molecular weights of "43 kDa (388aa) and 78 kDa (729aa)"

  • Observed molecular weight typically 80-90 kDa

  • Tissue-specific expression patterns

• Technical considerations:

  • Sample preparation issues (proteolysis during extraction)

  • Non-specific antibody binding

  • Cross-reactivity with RanBP10

Validation strategies include:

• Using phosphatase treatment to eliminate phosphorylation-dependent bands

• Comparing patterns across multiple antibodies recognizing different epitopes

• RanBP9 knockout/knockdown controls to identify specific bands

• Peptide competition assays to confirm specificity

How should I interpret changes in RanBP9 localization patterns in response to cellular stress?

RanBP9 shows complex subcellular distribution patterns that change in response to cellular stressors:

• Baseline localization:

  • RanBP9 shows "subcellular location in Cytoplasm, Nucleus, Cell membrane"

  • Distribution varies across cell types and physiological conditions

  • May form discrete puncta or diffuse patterns

• Stress-induced changes:

  • DNA damage may alter nuclear/cytoplasmic distribution

  • Phosphorylation by ATM could regulate localization after IR exposure

  • Changes may reflect functional adaptations to cellular stress

• Analysis methodology:

  • Quantify nuclear/cytoplasmic ratios using image analysis software

  • Perform subcellular fractionation followed by Western blotting

  • Consider co-localization with compartment-specific markers

• Interpretation framework:

  • Relocalization may indicate engagement in different protein complexes

  • Changes could reflect altered function in stress response pathways

  • Compare with known interactors (CTLH complex members, APP, ATM)

To establish causality, use phospho-mutants, nuclear localization signal (NLS) or nuclear export signal (NES) mutants, and specific stress pathway inhibitors to determine the mechanisms driving localization changes.

How can I optimize immunoprecipitation protocols to improve RanBP9 protein interaction studies?

Successful RanBP9 immunoprecipitation requires careful optimization:

• Lysis conditions:

  • Buffer composition significantly impacts interaction preservation

  • Include protease and phosphatase inhibitors

  • Adjust salt and detergent concentrations based on interaction strength

• IP antibody selection:

  • Choose antibodies that don't interfere with interaction domains

  • Consider using tagged RanBP9 (V5-HA) for efficient pulldown

  • Pre-clear lysates to reduce non-specific binding

• Cross-linking strategies:

  • For transient interactions, mild cross-linking can preserve complexes

  • IP-MS/MS experiments benefit from comparing cross-linked and non-cross-linked samples

  • Adjust cross-linker concentration and reaction time carefully

• Washing stringency:

  • Balance between preserving specific interactions and reducing background

  • Step-gradient washes can help determine interaction strength

  • Different wash buffers for different interaction types

• Validation approaches:

  • Perform reciprocal co-IPs

  • Include IgG control and input samples

  • Consider size exclusion chromatography as complementary approach

A proven methodology used monoclonal αHA-Agarose antibody with "1 mg of total protein extracts pre-cleared by incubating with Protein A/G Plus Agarose for 1 hr at 4°C" followed by overnight incubation with primary antibody .

What approaches can resolve conflicting data from different RanBP9 antibodies?

When facing discrepancies between different RanBP9 antibodies:

• Systematic comparison:

  • Test multiple antibodies side-by-side on the same samples

  • Map epitopes recognized by each antibody

  • Compare with manufacturer's validation data

• Validation using genetic approaches:

  • RanBP9 knockdown/knockout samples as gold-standard controls

  • Overexpression systems to confirm sensitivity

  • Tagged RanBP9 constructs as reference points

• Methodological optimization:

  • Test different sample preparation methods

  • Adjust detection systems (chemiluminescence vs. fluorescence)

  • Optimize blocking conditions to reduce non-specific binding

• Alternative approaches:

  • Mass spectrometry for unambiguous protein identification

  • Tagged mouse models like RanBP9-TT where "the double tag does not interfere with the essential functions of RanBP9"

  • Functional assays to support protein identification

The scientific literature acknowledges that "objective difficulties impair the investigation of cellular pathways and mechanisms in which RANBP9 takes part" due to antibody limitations , making thorough validation essential.

How can RanBP9 antibodies be used to investigate neurodegenerative disease mechanisms?

RanBP9 plays significant roles in neurodegenerative pathways that can be investigated using appropriate antibodies:

• APP processing and Aβ generation:

  • RanBP9 "markedly enhanced the levels of Aβ in the conditioned medium"

  • In transgenic mice, RanBP9 overexpression caused ">2-fold increase in Aβ40 levels"

  • Study protein interactions with APP, LRP, and BACE1 in disease models

• Synaptic protein regulation:

  • "RanBP9 overexpression significantly decreased the levels of synaptophysin and PSD-95 proteins"

  • "RanBP9-null mice showed increased levels of synaptophysin, PSD-95, and drebrin A protein levels"

  • Investigate mechanisms through co-IP and localization studies

• Proteolytic processing:

  • "RanBP9-N60, a 60-kDa proteolytically derived species of RanBP9... was increased 6-fold in the brains of patients with AD"

  • Track this fragment using antibodies against different epitopes

  • Investigate enzymes responsible for this processing

• Experimental approaches:

  • Compare RanBP9 expression/localization in control vs. disease tissues

  • Analyze age-dependent changes in animal models

  • Correlate with disease markers (amyloid plaques, tau tangles)

Given that "loss of synapses is the best pathological correlate of cognitive deficits in Alzheimer's disease," RanBP9's effects on synaptic proteins make it a "compelling" target for AD research .

What methodologies can reveal RanBP9's role in the ubiquitin-proteasome pathway?

As a core component of the CTLH E3 ubiquitin-protein ligase complex, RanBP9's role in protein degradation can be studied using:

• CTLH complex analysis:

  • Immunoprecipitate RanBP9 to co-purify the complex

  • All 11 members can be detected by IP-MS/MS within "the top 20 hits"

  • Map domain requirements for complex assembly

• E3 ligase activity assays:

  • In vitro ubiquitination assays with reconstituted components

  • Cell-based ubiquitination assays with tagged ubiquitin

  • The CTLH complex "selectively accepts ubiquitin from UBE2H"

• Substrate identification:

  • IP-MS/MS to identify proteins enriched after proteasome inhibition

  • Validation of known substrate HBP1 transcription factor

  • Confirmation using in vivo ubiquitination assays

• Structure-function analysis:

  • Domain mapping to identify regions required for E3 ligase activity

  • Create deletion mutants affecting specific protein interactions

  • Analyze effects on substrate recognition and ubiquitination

This research direction is particularly promising as "RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions" , extending their significance beyond neurodegeneration.

How can post-translational modifications of RanBP9 be studied effectively?

RanBP9 undergoes various post-translational modifications that affect its function:

• Phosphorylation analysis:

  • ATM phosphorylates RanBP9 at "S181 and S603 following IR exposure"

  • Generate phospho-specific antibodies against these sites

  • Compare phosphorylation patterns across different stimuli

• Mass spectrometry approaches:

  • Immunoprecipitate RanBP9 and analyze by MS/MS

  • Use titanium dioxide enrichment for phosphopeptides

  • Compare modification profiles across different conditions

• Functional analysis using mutants:

  • Create phospho-mimetic (S→D/E) and phospho-deficient (S→A) mutants

  • Analyze effects on protein interactions, localization, and function

  • Reconstitution experiments in knockout backgrounds

• Dynamics and regulation:

  • Time-course analysis after specific stimuli

  • Identify enzymes responsible (kinases, phosphatases, etc.)

  • Investigate cross-talk between different modifications

These studies will reveal how post-translational modifications regulate RanBP9's diverse functions in protein complex assembly, APP processing, and DNA damage response.

What experimental approaches can identify novel RanBP9 interacting partners in specific cell types?

The identification of cell type-specific RanBP9 interactors requires sophisticated approaches:

• Advanced mouse models:

  • The RanBP9-TurnX model enables "stringent biochemical studies at cell type specific level"

  • "RanBP9-3xHA can be turned into RanBP9-V5 tagged upon Cre-mediated recombination"

  • When crossed with LysM-Cre mice, reveals macrophage-specific interactions

• Proximity labeling techniques:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • APEX2 for temporal control of labeling

  • Compare interactomes across cell types

• IP-MS/MS optimization:

  • Cross-linking to capture transient interactions

  • Subcellular fractionation before IP (nuclear vs. cytoplasmic)

  • Advanced controls to filter out non-specific interactions

• Validation strategies:

  • Reciprocal co-IPs of identified partners

  • Co-localization studies using immunofluorescence

  • Functional validation through knockdown/knockout approaches

This approach has revealed that "the lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function" .