RNF128 Antibodies are employed in molecular biology techniques to study protein expression, interactions, and subcellular localization:
Western Blotting (WB): Quantifies RNF128 levels in lysates (e.g., tumor tissues vs. normal tissues).
Immunoprecipitation (IP): Identifies RNF128-binding partners (e.g., MPO in neutrophils , S100A8 in macrophages ).
Immunohistochemistry (IHC): Visualizes RNF128 in tissue sections (e.g., human liver cancer ).
ELISA: Measures soluble RNF128 in biological fluids (e.g., serum or culture supernatants).
RNF128 Antibodies have elucidated the protein’s immunomodulatory functions:
Acute Lung Injury: RNF128 binds myeloperoxidase (MPO) in neutrophils, inhibiting its activity and reducing inflammation .
Colorectal Cancer: High RNF128 expression correlates with tumor aggressiveness by suppressing the Hippo pathway .
Atherosclerosis: RNF128 stabilizes scavenger receptor B1 (SRB1), promoting foam cell formation and plaque progression .
E3 Ligase Activity: RNF128 ubiquitinates targets (e.g., MST in colorectal cancer , S100A8 in macrophages ) to regulate signaling pathways.
Subcellular Localization: Found in endosomes and lysosomes, where it interacts with membrane proteins like CD3 and SRB1 .
RNF128 (Ring Finger Protein 128), also known as GRAIL (Gene Related to Anergy in Lymphocytes), is an E3 ubiquitin ligase that catalyzes the formation of both 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains. This 428-amino acid protein has a molecular mass of approximately 46,521 daltons, though endogenous RNF128 is typically detected at around 66 kDa, likely due to post-translational modifications .
RNF128 functions primarily as an inhibitor of cytokine gene transcription, particularly IL2 and IL4, playing a crucial role in inducing and maintaining T-lymphocyte anergy - a state of unresponsiveness to antigenic stimulation . The protein ubiquitinates various targets including ARPC5 with 'Lys-48' linkages and COR1A with 'Lys-63' linkages, leading to their degradation and subsequent impairment of lamellipodium formation and reduced F-actin accumulation at the immunological synapse . Recent research has also revealed its role in the ubiquitination of SRB1, promoting its recycling to the cell membrane through a Rab11-dependent pathway .
RNF128 has a complex structural organization consisting of several functional domains. The protein contains:
A signal peptide at the N-terminus, essential for proper protein targeting and transport
A protease-associated (PA) domain that is evolutionarily conserved and responsible for capturing target proteins for cytosolic ubiquitination
A transmembrane domain
A RING finger domain at the C-terminus that confers E3 ubiquitin ligase activity
The N-terminal region, particularly the PA domain, is critical for protein-protein interactions. For instance, the PA domain specifically binds to SRB1, mediating their interaction . Research using truncated forms of RNF128 (RNF128-∆R, RNF128-∆SP, RNF128-N, RNF128-∆C, RNF128-C, and RNF128-∆N) has demonstrated that the N-terminus is required for binding to SRB1, while constructs lacking this region lose this ability .
Multiple suppliers offer anti-RNF128/GRAIL antibodies with various applications and specificities. These antibodies are available as:
Polyclonal antibodies (e.g., rabbit polyclonal) that recognize multiple epitopes within the protein
Antibodies that target specific regions such as the central portion of RNF128
Unconjugated antibodies for flexibility in detection methods
Most commercial antibodies are validated for applications including Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF), ELISA, and Immunoprecipitation (IP) . These antibodies demonstrate reactivity against human, mouse, and rat samples, with some also cross-reacting with other species .
For optimal Western blotting results with RNF128 antibodies, researchers should consider the following methodological approach:
Sample preparation: Due to RNF128's membrane association and post-translational modifications, use strong lysis buffers containing both ionic and non-ionic detergents (e.g., RIPA buffer supplemented with 0.1% SDS).
Antibody selection: Choose antibodies validated specifically for Western blotting. For example, Cell Signaling Technology's RNF128 antibody (#71590) is recommended at a 1:1000 dilution for this application .
Expected molecular weight: Look for bands between 60-80 kDa when detecting endogenous RNF128, rather than the calculated 46.5 kDa, as the protein undergoes extensive glycosylation and other post-translational modifications .
Controls: Include positive controls such as T-cell lysates where RNF128 is known to be expressed, and consider using RNF128 knockout/knockdown samples as negative controls to confirm antibody specificity.
Blocking and washing optimization: Use 5% BSA in TBST rather than milk for blocking, as phosphorylated epitopes may not be properly recognized in milk-based blocking solutions.
When performing immunoprecipitation of RNF128:
Antibody selection: Use antibodies specifically validated for immunoprecipitation, such as Cell Signaling Technology's RNF128 antibody (#71590) at a recommended dilution of 1:50 .
Lysis conditions: Employ gentler lysis buffers (compared to Western blotting) containing 1% NP-40 or Triton X-100 with protease and phosphatase inhibitors to preserve protein-protein interactions.
Pre-clearing step: Include a pre-clearing step with protein A/G beads to reduce non-specific binding.
Co-immunoprecipitation considerations: For studying RNF128's interactions with binding partners like SRB1, consider using epitope-tagged constructs (e.g., Flag-tagged RNF128 fragments and Myc-SRB1) to differentiate between the proteins in subsequent analyses .
Elution method: For detecting ubiquitination activity, consider using denaturing conditions (SDS or heat) for elution to disrupt protein-protein interactions and reveal all ubiquitinated species.
Rigorous validation of RNF128 antibody specificity requires multiple controls:
Genetic controls: Include RNF128 knockout/knockdown samples alongside wild-type samples. The absence or reduction of signal in knockout/knockdown samples confirms antibody specificity.
Overexpression controls: Compare endogenous expression with samples overexpressing RNF128 to verify that the antibody detects increased levels of the target protein.
Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application to samples. If specific, the antibody's binding to RNF128 should be blocked by the peptide, resulting in signal loss.
Cross-reactivity assessment: Test the antibody against related E3 ligases to ensure it doesn't cross-react with structurally similar proteins.
Multiple antibody validation: Compare results using antibodies from different suppliers or those targeting different epitopes of RNF128 to confirm consistent detection patterns.
RNF128 antibodies can be powerful tools for investigating ubiquitination dynamics through these advanced approaches:
Sequential immunoprecipitation: First immunoprecipitate RNF128 using specific antibodies, then probe for ubiquitinated substrates, or vice versa. This approach helps identify novel RNF128 substrates and characterize ubiquitination patterns.
Ubiquitin linkage-specific antibodies: Combine RNF128 antibodies with antibodies specific for K48- or K63-linked ubiquitin chains to distinguish between degradative and non-degradative ubiquitination. This is particularly relevant as RNF128 has been shown to catalyze both 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains .
Pulse-chase ubiquitination assays: Use RNF128 antibodies in conjunction with metabolic labeling of ubiquitin to track temporal changes in substrate ubiquitination. This can reveal how RNF128-mediated ubiquitination affects protein turnover or trafficking.
Proximity-based labeling: Combine RNF128 antibodies with techniques like BioID or APEX to identify proteins that associate with RNF128 in living cells, potentially revealing new substrates.
Subcellular fractionation: Use RNF128 antibodies on different cellular fractions to determine where RNF128-mediated ubiquitination predominantly occurs, correlating with its known cytoplasmic localization .
When encountering differences between the calculated (46.5 kDa) and observed (60-80 kDa) molecular weights of RNF128, researchers should consider these methodological approaches:
Deglycosylation experiments: Treat samples with glycosidases (PNGase F for N-linked glycans or O-glycosidase for O-linked glycans) prior to Western blotting. A shift in molecular weight after treatment confirms the presence of glycosylation, which has been reported for RNF128 .
Phosphatase treatment: Apply lambda phosphatase to determine if phosphorylation contributes to the higher observed molecular weight.
2D gel electrophoresis: Separate RNF128 by both isoelectric point and molecular weight to identify different post-translationally modified forms.
Mass spectrometry analysis: Perform mass spectrometry on immunoprecipitated RNF128 to precisely identify modifications and calculate their contribution to the observed molecular weight.
Expression of recombinant fragments: Compare the migration patterns of truncated RNF128 constructs (such as RNF128-∆R, RNF128-∆SP, RNF128-N, RNF128-∆C) against full-length protein to identify regions responsible for the molecular weight shift .
Recent research has revealed RNF128's involvement in membrane protein trafficking, particularly in recycling SRB1 to the plasma membrane . To further investigate this function:
Surface biotinylation assays: Combine RNF128 antibodies with surface biotinylation techniques to quantify plasma membrane proteins. After oxLDL treatment, biotinylate intact cells, perform avidin affinity purification, and analyze membrane protein levels by Western blotting with RNF128 antibodies .
Co-localization studies: Use immunofluorescence with RNF128 antibodies alongside markers for different cellular compartments (e.g., LAMP2 for lysosomes) to track protein trafficking. Quantitative analysis of co-localization can reveal how RNF128 affects the subcellular distribution of its substrates .
Live cell imaging: Combine fluorescently tagged RNF128 with total internal reflection fluorescence (TIRF) microscopy to observe real-time trafficking events at the plasma membrane.
Recycling assays: Implement antibody-based recycling assays where surface proteins are labeled with antibodies, allowed to internalize, and then recycled proteins are detected with secondary antibodies. Compare recycling rates in the presence and absence of RNF128.
Rab GTPase co-immunoprecipitation: As RNF128 has been shown to work with Rab11 in protein recycling, perform co-immunoprecipitation experiments with RNF128 antibodies to pull down Rab GTPases and identify additional trafficking machinery involved .
When selecting methods for RNF128 detection, researchers should consider these comparative advantages:
| Detection Method | Sensitivity | Specificity | Best Applications | Limitations |
|---|---|---|---|---|
| Western Blotting | Moderate | High when validated | Protein expression levels, molecular weight verification | Semi-quantitative, requires cell lysis |
| Immunohistochemistry | Moderate | Variable | Tissue distribution, cellular localization | Fixation artifacts, limited quantification |
| Immunofluorescence | High | Good with proper controls | Subcellular localization, co-localization studies | Background fluorescence issues |
| ELISA | Very high | Variable | Quantitative measurement in solution | Limited information on protein size or modifications |
| Immunoprecipitation | Moderate-High | High with validated antibodies | Protein-protein interactions, enrichment for PTM analysis | Requires optimization of lysis conditions |
| Flow Cytometry | High | Good with proper controls | Single-cell analysis, intracellular staining | Limited spatial information |
Multiple suppliers offer RNF128 antibodies validated for specific applications. For example, Novus Biologicals offers antibodies validated for WB, ICC, IF, IHC, and IHC-p, while Cell Signaling Technology's antibody is specifically validated for WB and IP applications .
To resolve contradictory findings in RNF128 research:
Domain-specific functional studies: Use the truncated forms of RNF128 (RNF128-∆R, RNF128-∆SP, RNF128-N, etc.) to systematically analyze which domains are responsible for specific functions . This approach can clarify whether contradictory results stem from different functional domains being studied.
Cell type-specific analysis: Compare RNF128 function across different cell types (e.g., T-cells vs. macrophages) using the same experimental conditions and antibodies. RNF128's function may vary significantly depending on cellular context.
Stimulus-dependent studies: Examine RNF128's role under different stimulation conditions (e.g., with or without oxLDL in macrophages) to determine if contradictory findings result from different cellular activation states .
Time-course experiments: Implement temporal analysis of RNF128 function to determine if contradictory findings stem from examining different time points in dynamic processes like protein trafficking or degradation.
Combined loss-of-function and rescue experiments: Generate RNF128 knockout models followed by reintroduction of wild-type or mutant RNF128 to precisely define functional domains and activities that may have been conflated in previous studies.
Researchers frequently encounter these challenges when working with RNF128 antibodies:
Inconsistent molecular weight detection: As discussed earlier, RNF128 appears at different molecular weights (46-80 kDa) due to post-translational modifications . Use positive controls with known RNF128 expression and consider deglycosylation experiments to standardize detection.
Background bands: Some RNF128 antibodies may detect non-specific bands. For example, Cell Signaling Technology's antibody (#71590) detects a 40 kDa band of unknown origin in some cell lines . Compare multiple antibodies targeting different epitopes and include knockout/knockdown controls to distinguish specific from non-specific signals.
Limited cross-reactivity: While some antibodies react with multiple species (human, mouse, rat), others have more limited reactivity . Carefully select antibodies validated for your species of interest and consider testing multiple antibodies if working with non-standard research models.
Epitope masking: Post-translational modifications or protein-protein interactions may mask antibody epitopes. Try multiple antibodies targeting different regions of RNF128 or modify extraction conditions to expose hidden epitopes.
Fixation sensitivity in microscopy: For immunofluorescence or immunohistochemistry, test different fixation methods (paraformaldehyde, methanol, acetone) as they can significantly affect epitope accessibility.
Different cell types may require specific optimization strategies for RNF128 detection:
T-cells and immune cells: As RNF128 plays a role in T-cell anergy, expression levels may vary dramatically based on activation state. Compare resting vs. activated T-cells and optimize lysis buffers to extract membrane-associated RNF128 efficiently .
Macrophages: Given RNF128's role in oxLDL-stimulated macrophages, consider optimization of stimulation times and concentrations when studying RNF128 in these cells .
Transfected cell lines: For overexpression studies, carefully titrate transfection amounts, as excessive RNF128 expression may lead to non-physiological interactions or subcellular localization.
Primary tissues: When analyzing RNF128 in tissue samples, optimize antigen retrieval methods for immunohistochemistry, as the protein's membrane association and modifications may require specific retrieval conditions.
Cellular compartment analysis: If studying RNF128's subcellular distribution, optimize fractionation protocols to effectively separate cytoplasmic, membrane, and organelle fractions without cross-contamination.
Several cutting-edge technologies hold promise for advancing RNF128 research:
Proximity labeling proteomics: BioID or TurboID fusion with RNF128 could identify transient interaction partners and substrates in living cells, potentially revealing novel functions.
Single-molecule tracking: Combining super-resolution microscopy with fluorescently labeled RNF128 antibodies could reveal dynamic interactions with substrates and trafficking machinery at the nanoscale level.
CRISPR-based screens: Genome-wide CRISPR screens in the context of RNF128 overexpression or knockout could identify synthetic lethal interactions and new functional pathways.
Ubiquitin-specific proteomics: Di-Gly remnant profiling combined with RNF128 manipulation could systematically identify all substrates of RNF128-mediated ubiquitination.
Cryo-EM structural studies: Determining the structure of RNF128 in complex with its substrates could provide mechanistic insights into how its PA domain recognizes specific targets and how its RING domain facilitates ubiquitin transfer.
RNF128 antibodies can be valuable tools for investigating disease mechanisms:
Autoimmunity: Given RNF128's role in T-cell anergy, analyze its expression and function in autoimmune disease samples to determine if dysregulation contributes to pathological immune activation .
Cardiovascular disease: Investigate RNF128's role in lipid metabolism and atherosclerosis by examining its regulation of SRB1 in cardiovascular disease models .
Cancer immunotherapy: Study how RNF128 affects T-cell responses in the tumor microenvironment, potentially identifying it as a target to enhance immunotherapy efficacy.
Neurodegenerative disorders: Explore RNF128's function in protein quality control and degradation in the context of protein aggregation diseases.
Biomarker development: Evaluate RNF128 expression patterns in patient samples to determine its potential as a diagnostic or prognostic biomarker for immune-related disorders.