RILP Antibody, FITC conjugated
Description
Production and Conjugation Methodology
FITC conjugation to RILP antibodies involves covalent coupling of the fluorophore to primary amine groups (e.g., lysine residues) . Key steps include:
Optimal Conjugation Parameters
Sodium azide must be removed pre-conjugation to prevent FITC inactivation . Commercial RILP antibodies (e.g., Proteintech’s 13574-1-AP or 82996-4-PBS) are often unconjugated but can be labeled in-house .
Applications in Research
RILP-FITC antibodies are used to study:
Lysosomal Dynamics and Autophagy
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Rab7-RILP Interaction: FITC-conjugated RILP antibodies enable visualization of Rab7-RILP complexes during autophagosome-lysosome fusion .
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Lysosome Localization: Co-localization studies with markers like LAMP1 or LC3 using FITC-RILP antibodies confirm lysosomal compartmentalization .
Immunofluorescence and Flow Cytometry
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Cellular Staining: FITC-RILP antibodies detect endogenous RILP in permeabilized cells, with optimal dilutions typically 1:500–1:1000 .
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Multiplex Imaging: Conjugation-ready RILP antibodies (e.g., 82996-4-PBS) allow custom FITC labeling for compatibility with other fluorophores .
Western Blotting (WB)
While FITC is not commonly used in WB, unconjugated RILP antibodies (e.g., ab140188) are validated for WB and immunoprecipitation (IP) .
Key Considerations
Case Study: In autophagy studies, IGF-I treatment restored Rab7-RILP interaction, detected via GST pull-down assays and immunoblotting . FITC-conjugated antibodies could enhance real-time visualization of this interaction.
Rab7-RILP Pathway Insights
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Autophagy Regulation: Rab7-RILP interaction is critical for autophagosome-lysosome fusion; IGF-I/Akt signaling restores this interaction during trophic factor withdrawal .
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Lysosomal Morphology: RILP depletion disrupts lysosome distribution, impairing cellular homeostasis .
Challenges in Conjugation
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Rab effector protein involved in late endocytic transport to lysosomes and other degradative compartments. It plays a crucial role in regulating lysosomal morphology and distribution. RILP induces the recruitment of dynein-dynactin motor complexes to Rab7A-positive late endosomes and lysosomes, facilitating the centripetal movement of phagosomes and their fusion with late endosomes and lysosomes.
RILP's function is implicated in several key processes. Research highlights its involvement in:
- Hepatitis C Virus (HCV) Infection: HCV alters cellular trafficking by cleaving RILP, redirecting Rab7-containing vesicles to kinesin-dependent transport and enhancing virion secretion. PMID: 27791088
- Breast Cancer Cell Invasion: RILP inhibits breast cancer cell invasion by interacting with RalGDS, suppressing its guanine nucleotide exchange factor activity for RalA. PMID: 26469971
- Vacuolar ATPase Regulation: RILP regulates vacuolar ATPase activity through interaction with the V1G1 subunit. PMID: 26180254, PMID: 24762812
- Interaction with the HOPS Complex: The VPS41 subunit of the HOPS complex is a major interacting partner of RILP. PMID: 25445562
- Dynein Motor Interaction and Cholesterol Sensing: RILP interacts with both the HOPS complex and the p150(Glued) subunit of the dynein motor. ORP1L acts as a cholesterol-sensing switch, modulating these interactions. PMID: 23729732
- Regulation of Lysosomal Morphology: A unique region within RILP is responsible for its role in regulating lysosomal morphology and its interaction with Rab7 and Rab34. PMID: 14668488
- Rab7 Interaction: The crystal structure of the Rab7-RILP complex reveals specific interactions between Rab7 and RILP. PMID: 15933719
- Self-Association: RILP exhibits self-association, likely forming homodimers. PMID: 15996637
- Post-translational Modifications: RILP undergoes prenylation and phosphorylation at two protein kinase A sites. PMID: 16417580
- Vesicle Fusion and Transport: RILP plays a role in regulating vesicle fusion and transport mediated by Rab7. PMID: 16631113
- ESCRT-II Complex Interaction: RILP interacts with the EAP30/SNF8/VPS22 subunit of the ESCRT-II complex. PMID: 16857164
- Multivesicular Endosome Biogenesis and EGFR Trafficking: RILP is required for multivesicular endosome biogenesis and the degradative trafficking of EGFRs. PMID: 17959629
- REST/NRSF Nuclear Translocation: RILP forms a complex with REST/NRSF, dynactin p150(Glued), huntingtin, HAP1, involved in REST/NRSF nuclear translocation. PMID: 18922795
- Late Endosome Positioning: ORP1L's interaction with VAP influences Rab7-RILP-p150 Glued interactions and late endosome positioning. PMID: 19564404
