RILP Antibody

What is RILP and what is its function in cellular processes?

RILP is a 45 kDa protein that specifically binds to Rab7GTP at its C-terminus. It contains a domain comprising two coiled-coil regions typical of myosin-like proteins and is found mainly in the cytosol. RILP plays a crucial role in controlling transport to endocytic degradative compartments through its interaction with Rab7. It can be efficiently recruited to late endosomal and lysosomal membranes by Rab7GTP, and expression of RILP can reverse or prevent the effects of Rab7 dominant-negative mutants. Functionally, RILP-C33 (a truncated form lacking the N-terminal half) strongly inhibits epidermal growth factor and low-density lipoprotein degradation, demonstrating RILP's importance in degradative pathways .

What applications can RILP antibody be used for in laboratory research?

RILP antibody (such as the commercially available 13574-1-AP) can be applied in multiple experimental techniques with specific protocols for each application:

Each application requires specific protocols and optimization for best results .

What is the recommended dilution for RILP antibody in different experimental applications?

Based on validated protocols, the following dilution ranges are recommended for RILP antibody (13574-1-AP):

It is strongly recommended that researchers titrate the antibody in their specific experimental systems to obtain optimal results, as the ideal dilution may vary depending on sample type, detection method, and experimental conditions .

What is the difference between RILP and Rilp-like proteins (Rilpl1 and Rilpl2)?

While RILP primarily functions in endolysosomal trafficking through Rab7 interaction, Rilp-like proteins (Rilpl1 and Rilpl2) have distinct functions in ciliary regulation. Specifically, Rilpl1 and Rilpl2 regulate ciliary membrane protein concentration by promoting protein removal from the primary cilium. Immunofluorescence studies of differentiating MTEC (Mouse Tracheal Epithelial Cell) cultures show that endogenous Rilpl2 localizes to the apical surface of a subset of cells, consistent with its role in ciliary function. These proteins share structural similarities but have evolved specialized roles in different cellular compartments .

How can I validate the specificity of RILP antibody in my experimental system?

Thorough validation of RILP antibody specificity should include multiple complementary approaches:

• Western blot analysis: The antibody should detect a band at 41-45 kDa in appropriate cell lysates. RILP protein has been detected in various cell lines including HeLa, CaCo2, MKN28, FEUN, 293 cells, and peripheral blood lymphocytes .

• Immunofluorescence with known markers: Confirm co-localization with late endosomal/lysosomal markers (Lamp1, Lamp2, CathD) and absence of co-localization with markers of other compartments (adaptin γ, transferrin receptor, EEA1, PDI) .

• RNA interference: Use siRNA/shRNA targeting RILP to confirm signal reduction. The literature describes shRNA constructs against RILP (pSilencer-RILP-st1) that can be used for this purpose .

• Overexpression controls: Transfection with RILP constructs should result in increased signal intensity in a pattern consistent with late endosomal/lysosomal localization .

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining.

What are the optimal fixation and permeabilization methods for RILP immunofluorescence?

Visualization of endogenous RILP requires careful consideration of fixation and permeabilization methods:

• For membrane-associated RILP: Permeabilization with saponin before fixation helps wash out excess cytosolic proteins, allowing better visualization of the membrane-associated fraction. This approach successfully reveals co-localization between RILP and late endosomal/lysosomal markers in CaCo2 cells .

• For overexpressed RILP: Standard fixation protocols (typically paraformaldehyde fixation followed by detergent permeabilization) are usually sufficient as the proteins show clear co-localization with Lamp1 .

• Antigen retrieval for tissue sections: For IHC applications, TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may also be used as an alternative .

• Detailed protocols: Follow manufacturer-recommended protocols as starting points, then optimize based on your specific experimental system .

What controls should I include when working with RILP antibody?

A comprehensive set of controls is essential for proper interpretation of RILP antibody experiments:

• Positive controls:

  • Cell lines with confirmed RILP expression: HEK-293 and HepG2 cells for Western blot

  • Tissues: Human kidney tissue or mouse brain tissue for IHC

  • Cell lines for IF/ICC: HepG2 cells

• Negative controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG at equivalent concentration)

  • Pre-immune serum (when available)

  • RILP knockdown samples

• Specificity controls:

  • Peptide competition assay

  • Parallel detection with another RILP antibody targeting a different epitope

  • RILP-null cells or tissues (if available)

• Localization controls:

  • Co-staining with established markers (Lamp1 for late endosomes/lysosomes should show co-localization, while EEA1 for early endosomes should not)

How can I use RILP antibody to study Rab7-dependent endolysosomal trafficking?

RILP antibody serves as a powerful tool for investigating Rab7-dependent trafficking pathways:

• Membrane recruitment assays: The search results show that active Rab7 (Rab7Q67L) can recruit RILP to membranes, while inactive Rab7 (Rab7T22N) cannot . By expressing these Rab7 mutants and staining for endogenous RILP, researchers can assess Rab7 activity states in different cellular contexts.

• Co-localization studies: RILP antibody can be used alongside Rab7 antibody to assess their co-localization under different experimental conditions or treatments, revealing potential disruptions in Rab7-RILP interactions .

• Trafficking assays: Since RILP is crucial for degradative pathways, RILP antibody can be used to monitor the effects of various treatments on RILP expression and localization in relation to cargo degradation. The truncated form RILP-C33 strongly inhibits degradation pathways, making it a useful tool in such studies .

• Pull-down experiments: The antibody can be used in immunoprecipitation to isolate RILP and its interacting partners, enabling investigation of how various conditions affect the Rab7-RILP interaction network.

• Quantitative analysis: Measuring co-localization coefficients between RILP and various endolysosomal markers after experimental perturbations can provide insights into trafficking defects.

What are the considerations when using RILP antibody in co-localization studies?

When designing co-localization experiments with RILP antibody, several factors require attention:

• Expected patterns: RILP shows high co-localization with late endosomal/lysosomal markers (Lamp1, Lamp2, CathD) but not with markers of other compartments (adaptin γ, transferrin receptor, EEA1, PDI) .

• Antibody compatibility: Since the RILP antibody 13574-1-AP is raised in rabbit , pair it with markers detected by antibodies raised in different species to avoid cross-reactivity of secondary antibodies.

• Sample preparation: For endogenous RILP, permeabilization with saponin before fixation helps visualize membrane-associated RILP and reduces cytosolic background .

• Signal intensity balance: RILP expression varies between cell types, with higher levels in CaCo2 cells compared to HeLa cells . Adjust exposure settings accordingly when comparing different cell lines.

• Resolution considerations: Standard confocal microscopy may not fully resolve closely associated but distinct structures. Consider super-resolution techniques for detailed co-localization analyses of endolysosomal compartments.

How can I use RILP antibody to investigate lysosomal positioning and function?

RILP antibody enables detailed investigation of lysosomal dynamics:

• Position analysis: RILP affects the distribution of late endosomal/lysosomal compartments . Use the antibody to track changes in lysosomal positioning under different experimental conditions.

• Rab7 activity correlation: Since RILP recruitment to lysosomes depends on active Rab7 , RILP staining patterns provide an indirect readout of Rab7 activity and its effect on lysosomal positioning.

• Motor protein interactions: RILP connects lysosomes to dynein-dynactin motor complexes. Combining RILP antibody with dynein/dynactin markers can reveal how this interaction affects lysosomal movement and positioning.

• Morphological analysis: Overexpression of RILP or its truncated form RILP-C33 strongly affects the morphology of the late endosomal/lysosomal compartment . The antibody can help distinguish between normal and altered lysosomal morphology.

• Functional correlation: Combine RILP antibody staining with functional lysosomal assays (pH indicators, degradation assays) to correlate RILP localization with lysosomal function.

Can RILP antibody be used to differentiate between active and inactive Rab7 pathways?

RILP antibody provides valuable insights into Rab7 activity states:

• Membrane association: Active Rab7 (Rab7Q67L) recruits RILP to membranes, while inactive Rab7 (Rab7T22N) does not . The degree of membrane-associated RILP detected by the antibody serves as an indirect measure of Rab7 activity.

• Subcellular distribution: In cells with active Rab7, RILP antibody shows stronger co-localization with late endosomal/lysosomal markers. In cells with inactive Rab7, RILP remains predominantly cytosolic .

• Experimental approach: When expressed alongside constitutively active (Rab7Q67L) or dominant-negative (Rab7T22N) Rab7 mutants, RILP antibody reveals how these activity states affect endogenous RILP distribution .

• Quantitative assessment: The ratio of membrane-bound to cytosolic RILP (measured by subcellular fractionation and Western blotting) provides a quantitative assessment of Rab7 activity in different experimental conditions.

• Limitations: This approach provides an indirect measurement of Rab7 activity and should be complemented with direct Rab7 activity assays when possible.

What are common issues when using RILP antibody and how can they be resolved?

Researchers may encounter several challenges when working with RILP antibody:

• High cytosolic background: RILP is predominantly cytosolic, which can mask membrane-associated signals. Solution: Permeabilize with saponin before fixation to wash out excess cytosolic proteins .

• Weak signal in immunofluorescence: This may result from low expression levels in certain cell types. Solution: Use CaCo2 cells as positive controls since they express higher levels of RILP . Optimize antibody concentration and detection systems.

• Non-specific bands in Western blot: Solution: Include positive controls (HEK-293, HepG2 cells) and optimize blocking and washing conditions.

• Variable results across cell types: RILP expression varies between cell lines . Solution: Validate the antibody in each cell line of interest and adjust protocols accordingly.

• Discrepancy between overexpression and endogenous patterns: Overexpressed RILP can alter the morphology of the late endosomal/lysosomal compartment . Solution: Use both approaches and carefully interpret differences in light of potential functional effects.

What are potential pitfalls when interpreting RILP antibody staining in cells with disrupted endolysosomal systems?

When studying systems with compromised endolysosomal function, several considerations are crucial:

• Altered subcellular distribution: Endolysosomal disruptions may redistribute RILP, making interpretation challenging. Always compare with control cells processed identically.

• Expression level variations: Endolysosomal stress can alter RILP expression, affecting signal intensity independently of localization changes. Complement immunofluorescence with Western blot quantification.

• Co-localization changes: Disrupted endolysosomes may show altered co-localization patterns between RILP and standard markers. Use multiple markers to build a comprehensive picture.

• Compensatory mechanisms: Cells may upregulate Rilp-like proteins (Rilpl1, Rilpl2) in response to endolysosomal disruption, potentially creating confounding signals with some antibodies.

• Fixation artifacts: Disrupted endolysosomes may be more sensitive to fixation procedures. Test multiple fixation methods and include appropriate controls.

How can RILP antibody be used in studies of RILP mutants or domain analysis?

The literature describes several RILP mutants (F222A, E226A, L231A, E233A, R234A, and N235A) that can be studied using RILP antibody:

• Expression verification: RILP antibody can confirm expression of mutant proteins at levels comparable to wild-type.

• Localization analysis: Compare subcellular distribution of mutants versus wild-type RILP to identify domains critical for proper localization.

• Functional correlation: Combine localization studies with functional assays to correlate specific domains with RILP functions.

• Interaction studies: Use co-immunoprecipitation with RILP antibody to investigate how mutations affect RILP's interactions with binding partners.

• Structure-function studies: For comprehensive domain analysis, combine deletion mutants, point mutants, and chimeric proteins, using the antibody to track their localization and function.

How can RILP antibody be used in studies involving RILP knockdown or overexpression?

RILP antibody is essential for manipulation studies:

• Knockdown verification: Confirm successful knockdown at the protein level using Western blot or immunofluorescence. The literature describes shRNA constructs targeting RILP (pSilencer-RILP-st1) .

• Overexpression confirmation: Verify successful overexpression of RILP or truncated forms like RILP-C33, ensuring expression at appropriate levels .

• Rescue experiments: In knockdown/knockout followed by rescue approaches, RILP antibody confirms expression of rescue constructs, especially those designed to be resistant to the knockdown strategy.

• Phenotypic analysis: Since RILP-C33 inhibits degradative pathways and causes lysosomal dispersion similar to Rab7 dominant-negative mutants , RILP antibody helps correlate RILP levels with these phenotypes.

• Mutant analysis: When studying mutant forms of RILP, the antibody confirms their expression levels are comparable to wild-type RILP, ensuring phenotypic differences are not due to expression disparities.