RIN3 Antibody, HRP conjugated

What is the optimal dilution range for RIN3 antibody, HRP conjugated in Western blot applications?

For Western blot applications, HRP-conjugated RIN3 antibodies typically require optimization between 1:1000 and 1:5000 dilutions in 5% BSA or non-fat milk in TBST. Begin with manufacturer-recommended dilutions, then adjust based on signal-to-noise ratio. For detecting endogenous RIN3 protein (approximately 105 kDa), a 1:2000 dilution often provides optimal results with 20-40 μg of total protein lysate. Always perform a dilution series (e.g., 1:1000, 1:2000, 1:5000) when working with new antibody lots to determine optimal conditions for your specific experimental system .

How should RIN3 antibody samples be stored to maintain HRP conjugate activity?

Proper storage is critical for maintaining HRP activity in conjugated antibodies. Store HRP-conjugated RIN3 antibodies at -20°C in small aliquots (20-50 μL) to minimize freeze-thaw cycles. Include a cryoprotectant such as 50% glycerol and avoid repeated freeze-thaw cycles (limit to ≤5). For short-term storage (1-2 weeks), refrigeration at 4°C is acceptable if the antibody contains preservatives. Never store diluted working solutions for extended periods, as this significantly reduces signal intensity. HRP activity typically decreases by approximately 10-15% after 6 months even under optimal storage conditions .

What controls should be included when using RIN3 antibody, HRP conjugated for the first time?

When using HRP-conjugated RIN3 antibodies in initial experiments, include the following controls:

• Positive control: Lysates from tissues/cells known to express RIN3 (e.g., hippocampal or cortical tissue, as RIN3 is upregulated in these regions in AD models)

• Negative control: Lysates from RIN3 knockout cells or tissues with minimal RIN3 expression

• Loading control: Probing for housekeeping proteins (β-actin, GAPDH) to ensure equal loading

• Peptide competition assay: Pre-incubating the antibody with immunizing peptide to confirm specificity

• Secondary antibody-only control: Omitting primary antibody to identify non-specific binding

This comprehensive approach enables validation of antibody specificity and ensures reliable interpretation of experimental results .

How does HRP conjugation affect RIN3 antibody performance in immunohistochemistry applications?

HRP conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibodies in immunohistochemistry (IHC). While this streamlines the protocol, it can impact antibody performance in several ways:

For optimal results in neuronal tissue samples, use antigen retrieval (10mM sodium citrate, pH 6.0, 95°C for 20 minutes) and extend primary antibody incubation to 36-48 hours at 4°C when detecting RIN3 in brain sections .

How can RIN3 antibody, HRP conjugated be used to investigate RIN3's interaction with other Alzheimer's Disease risk factors?

For investigating RIN3's interactions with BIN1 and CD2AP (known AD risk factors), combine immunoprecipitation with HRP-conjugated RIN3 antibody detection in a co-IP Western blot approach:

• Perform co-immunoprecipitation using anti-BIN1 or anti-CD2AP antibodies

• Separate proteins by SDS-PAGE and transfer to PVDF membranes

• Block membranes with 5% BSA in TBST for 1 hour

• Apply HRP-conjugated RIN3 antibody (1:2000) directly to membranes

• Wash and develop using enhanced chemiluminescence

This method enables direct detection of RIN3 in protein complexes without secondary antibody interference. Research has shown that RIN3 interacts with BIN1 via its N-terminal region (amino acids 1-586) and with CD2AP through its proline-rich domains (367-390aa, 445-462aa). These interactions recruit both proteins to Rab5-positive early endosomes, potentially impacting endosomal trafficking in AD pathogenesis .

What are the critical parameters for quantifying RIN3 upregulation in Alzheimer's Disease models using HRP-conjugated antibodies?

When quantifying RIN3 upregulation in AD models, several critical parameters must be controlled:

• Sample preparation: Homogenize tissue in RIPA buffer containing protease inhibitors, phosphatase inhibitors, and specific endosomal fraction enrichment if necessary

• Protein normalization: Use BCA assay for precise quantification; load 20-40 μg of total protein

• Membrane optimization: Use 0.45 μm PVDF membranes for optimal protein retention

• Exposure calibration: Generate a standard curve using recombinant RIN3 protein (0.1-10 ng)

• Region-specific analysis: Separately analyze hippocampus, cortex, and other relevant brain regions

Research has demonstrated significant RIN3 upregulation in APP/PS1 mouse hippocampus and cortex, correlating with enlarged Rab5-positive early endosomes in basal forebrain cholinergic neurons. Quantitative measurements show approximately 2.5-fold increase in RIN3 mRNA levels in these regions compared to age-matched controls .

How should experimental designs be modified when investigating RIN3's role in endosomal trafficking using HRP-conjugated antibodies?

When investigating RIN3's role in endosomal trafficking:

• Combine HRP-conjugated RIN3 antibody with fluorescently-labeled Rab5, Rab7, and Rab11 antibodies for multiplex analysis of endosomal compartments

• Implement subcellular fractionation to isolate early endosomes using sucrose gradient ultracentrifugation

• Use live-cell imaging with RIN3 overexpression/knockdown systems to track APP and BACE1 trafficking

• Establish rescue experiments with dominant-negative Rab5 (Rab5^S34N) to confirm Rab5-dependency

• Analyze axonal transport parameters (velocity, directionality, stationary periods) in primary neurons

Research has shown that RIN3 upregulation impairs APP and BACE1 trafficking by increasing stationary vesicles and reducing trafficking velocities. This ultimately leads to increased APP CTF production, a mechanism that can be reversed by Rab5^S34N expression, indicating Rab5 activation as the primary mechanism of RIN3-mediated endosomal dysfunction .

What are the most common causes of weak signal when using RIN3 antibody, HRP conjugated, and how can they be resolved?

When encountering weak signals with HRP-conjugated RIN3 antibodies, consider these common causes and solutions:

Additionally, signal amplification systems (SuperSignal West Femto) can increase sensitivity by 10-50 fold. Extended exposure times may be necessary as RIN3 is expressed at relatively low levels in some neural tissues under basal conditions .

How can cross-reactivity issues be addressed when studying RIN3 alongside other RIN family proteins?

The RIN protein family (RIN1, RIN2, RIN3) shares significant homology, potentially leading to cross-reactivity. To address this:

• Validate antibody specificity using overexpression systems for each RIN family member

• Perform peptide competition assays with specific peptides from each RIN protein

• Include RIN1/RIN2/RIN3 knockout samples as negative controls

• Use domain-specific antibodies targeting unique regions (RIN3's extended N-terminal region)

• Implement immunodepletion strategies to confirm signal specificity

RIN3 shares approximately 45% homology with RIN1 and 49% with RIN2, with highest conservation in the Vps9 and RA domains. Selecting antibodies targeting the more divergent SH2 domain (N-terminal) or internal regions can minimize cross-reactivity. Preabsorption with recombinant RIN1/RIN2 proteins can further enhance specificity for RIN3 detection .

What methodological modifications are needed when studying phosphorylated forms of RIN3 with HRP-conjugated antibodies?

When studying phosphorylated RIN3 forms:

• Include phosphatase inhibitors (50 mM NaF, 10 mM Na₃VO₄, 10 mM Na₄P₂O₇) in all lysis buffers

• Maintain samples at 4°C throughout processing to prevent dephosphorylation

• Use Phos-tag™ SDS-PAGE for enhanced separation of phosphorylated species

• Consider lambda phosphatase treatment of control samples to confirm phospho-specificity

• Use phospho-specific RIN3 antibodies alongside total RIN3 detection

Research indicates that RIN3 phosphorylation status affects its GEF activity toward Rab5. Western blotting for phosphorylated RIN3 requires careful sample handling, as phosphorylation sites can be rapidly dephosphorylated post-extraction. Comparing phosphorylated-to-total RIN3 ratios provides more reliable quantification than absolute phospho-signal alone .

How do immunofluorescence and HRP-based detection methods for RIN3 compare in neuronal cultures?

When studying RIN3 in neuronal cultures, researchers must choose between immunofluorescence and HRP-based detection methods:

What are the methodological considerations for studying the impact of RIN3 upregulation on Tau phosphorylation using HRP-conjugated antibodies?

When investigating RIN3's impact on Tau phosphorylation:

• Use site-specific phospho-Tau antibodies (pSer396/pSer404, PHF-1 epitope; pThr231, AT180 epitope)

• Implement sequential blotting protocols to detect total-Tau and phospho-Tau on the same membrane

• Include GSK3β and CDK5 activity assays to identify kinase involvement

• Perform RIN3 overexpression/knockdown in primary neurons with wild-type and mutant BIN1

• Use Rab5^S34N expression as a control to test Rab5-dependency

Research has demonstrated that upregulation of RIN3 increases phosphorylated Tau levels, an effect that can be rescued by dominant-negative Rab5 expression. This suggests that RIN3-induced Tau hyperphosphorylation occurs through Rab5 activation. The neuronal isoform of BIN1 appears to be required for this effect, indicating a complex RIN3-BIN1-Tau relationship that may contribute to AD pathogenesis .

How can RIN3 antibodies be used to investigate the RIN3 interactome in Alzheimer's Disease models?

For comprehensive analysis of the RIN3 interactome in AD models:

• Perform immunoprecipitation with RIN3 antibodies from brain tissue lysates

• Use crosslinking approaches (DSP, formaldehyde) to capture transient interactions

• Analyze immunoprecipitates by mass spectrometry (LC-MS/MS)

• Validate key interactions by reciprocal co-IP and proximity ligation assays

• Compare interactome between control and AD model tissues to identify disease-specific interactions

Previous research using flag-tagged RIN3 identified 380 interacting proteins, with significant enrichment for vesicular transport proteins. Key AD-relevant interactors include BIN1 (PSMs = 22, coverage = 38%) and CD2AP (PSMs = 21, coverage = 30%). Domain mapping experiments suggest that the SH2 or proline-rich domains of RIN3 are required for these interactions and for targeting these proteins to Rab5-positive early endosomes .

How can RIN3 antibodies be used to investigate the relationship between RIN3 upregulation and APP processing in neuronal models?

To investigate RIN3's role in APP processing:

• Combine HRP-conjugated RIN3 antibodies with antibodies against APP and its proteolytic products (βCTFs, αCTFs)

• Implement subcellular fractionation to isolate endosomal compartments and analyze APP fragment distribution

• Use RIN3 overexpression/knockdown systems with wild-type APP and AD-associated APP mutants

• Monitor BACE1 activity and localization in relation to RIN3 expression levels

• Analyze axonal transport of APP vesicles using live-cell imaging in primary neurons

Research has demonstrated that RIN3 overexpression promotes APP cleavage to increase carboxyl terminal fragments (CTFs), particularly βCTFs. This effect appears to be mediated through CD2AP interaction and can be rescued by dominant-negative Rab5 expression. The production of βCTFs is particularly significant as these fragments exhibit neuronal toxicity independent of Aβ and are linked to early cellular pathology in AD .

What methodological considerations are important when investigating RIN3's role in different cellular compartments using HRP-conjugated antibodies?

When studying RIN3's distribution across cellular compartments:

• Implement differential centrifugation to isolate membrane fractions, cytosol, and organelles

• Use sucrose gradient ultracentrifugation for further separation of early, late, and recycling endosomes

• Combine with organelle-specific markers (EEA1 for early endosomes, Rab7 for late endosomes)

• Consider detergent resistance when preparing samples (use 1% Triton X-100 and 0.5% SDS)

• Validate findings with immunofluorescence colocalization using confocal microscopy

Research indicates that RIN3 predominantly localizes to early endosomes (Rab5-positive) but not to late endosomes (Rab7-positive) or recycling endosomes (Rab11-positive). The SH2 and RH domains of RIN3 appear essential for this specific targeting, as deletion of either domain results in diffuse cytoplasmic localization of RIN3, BIN1, and CD2AP, without proper recruitment to early endosomes .

How should researchers approach quantitative analysis of RIN3 expression across different brain regions in Alzheimer's Disease models?

For quantitative analysis of region-specific RIN3 expression:

• Implement laser capture microdissection to isolate specific brain regions

• Use RT-qPCR with region-specific internal controls for mRNA quantification

• Perform Western blotting with HRP-conjugated RIN3 antibodies for protein quantification

• Calibrate using recombinant RIN3 protein standards (0.1-10 ng range)

• Normalize to region-specific housekeeping genes/proteins to account for cellular composition differences

Research has shown significant upregulation of RIN3 mRNA in the hippocampus and cortex of APP/PS1 mouse brains compared to wild-type controls. This upregulation correlates with early endosome enlargement in basal forebrain cholinergic neurons cultured from E18 APP/PS1 mouse embryos, suggesting region-specific vulnerability to RIN3-mediated endosomal dysfunction in AD pathogenesis .