RPL7 Antibody

What is RPL7 and what are its primary cellular functions?

RPL7 (Ribosomal Protein L7) is a component of the large ribosomal subunit (60S) that plays crucial roles in protein synthesis. It functions beyond structural support for ribosomes by serving regulatory roles in the translation apparatus. Specifically, RPL7 binds to G-rich structures in 28S rRNA and mRNAs and can inhibit cell-free translation of mRNAs, suggesting a regulatory function in protein synthesis . The protein interacts with other ribosomal proteins such as RPL13 and RPL10A, contributing to proper progression of translational pathways necessary for cell growth and differentiation . RPL7 is also known as uL30 in the systematic naming of ribosomal proteins, reflecting its evolutionary conservation and functional importance across species.

What applications have RPL7 antibodies been validated for in research?

RPL7 antibodies have been validated for multiple research applications, with strong evidence supporting their use in:

• Western blotting (WB) - Detecting RPL7 protein expression in cell and tissue lysates

• Immunoprecipitation (IP) - Isolating RPL7-containing protein complexes

• Immunohistochemistry on paraffin-embedded samples (IHC-P) - Localizing RPL7 in tissue sections

• Immunocytochemistry/Immunofluorescence (ICC/IF) - Visualizing RPL7 subcellular localization

Most commercially available antibodies have been rigorously tested in these applications, with documented specificity demonstrated through validation techniques such as siRNA knockdown controls. For instance, the RPL7 antibody ab72550 shows significant reduction in signal when RPL7 is depleted by siRNA in Western blot applications, confirming its specificity for the target protein .

What species reactivity can be expected with RPL7 antibodies?

Most commercially available RPL7 antibodies demonstrate cross-reactivity across multiple mammalian species due to the high conservation of ribosomal proteins throughout evolution. Based on the search results, RPL7 antibodies typically react with human and mouse samples with high specificity . Some antibodies also show reactivity with rat samples .

When selecting an RPL7 antibody for research, it's important to note that manufacturers classify reactivity into different categories:

• Tested and confirmed to work (backed by product promises)

• Expected to work based on sequence homology (may be covered by product guarantees)

• Predicted to work based on homology (may not be covered by guarantees)

For novel species applications, researchers should consider the degree of sequence homology in the immunogen region and may need to perform validation experiments before proceeding with full-scale studies.

How can RPL7 antibodies be utilized in ribosome-associated mRNA isolation studies?

RPL7 antibodies play a crucial role in methodologies designed to isolate cell-type-specific ribosome-associated mRNAs, particularly in heterogeneous tissues like brain or testis. One such approach is the RiboTag methodology, which utilizes epitope-tagged ribosomal proteins including RPL22-HA for immunoprecipitation of actively translating polyribosomes .

In this context, RPL7 antibodies serve two important functions:

• As validation tools: Western blot analysis using RPL7 antibodies confirms successful co-immunoprecipitation of intact ribosomal complexes. The presence of RPL7 in immunoprecipitates pulled down with anti-HA antibodies confirms that the tagged protein (RPL22-HA) is incorporated into functional ribosomes .

• For quality control: Detection of RPL7 in immunoprecipitated samples serves as evidence that complete ribosomal complexes have been isolated rather than just the tagged protein alone.

The methodology typically achieves approximately 25% efficiency in immunoprecipitating ribosomal complexes, regardless of whether the tagged cells constitute a large or small fraction of the total tissue, making it suitable for studying even rare cell populations .

Researchers should note that this approach offers significant advantages over alternatives like LCM (Laser Capture Microdissection) or FACS-based methods, as it avoids disruption of three-dimensional cellular relationships and preserves the in vivo cellular state, preventing artificial changes in gene expression that may occur during tissue processing .

What is the significance of RPL7/L12 immune response in colorectal cancer research?

Research has uncovered a compelling temporal association between humoral immune responses against bacterial ribosomal protein RPL7/L12 and colorectal cancer (CRC) progression. Studies have consistently demonstrated that:

• Polyp patients and early-stage (I/II) CRC patients exhibit significantly elevated serum anti-RPL7/L12 IgG levels compared to healthy controls .

• Late-stage CRC patients (stages III/IV with lymph node or distant metastasis) show reduced or normalized anti-RPL7/L12 antibody levels .

This pattern suggests a unique immunological response that occurs predominantly during early carcinogenesis rather than as a general consequence of compromised intestinal barrier function. Supporting this interpretation, the studies found no parallel increase in antibodies against endotoxin (a common bacterial cell wall component) .

The significance for researchers is multi-faceted:

• Diagnostic potential: The elevated anti-RPL7/L12 response may serve as a biomarker for early-stage CRC detection.

• Mechanistic insights: The data supports a temporal association between Streptococcus bovis exposure and colorectal carcinogenesis, where bacterial antigens may play a role in early disease stages.

• Geographical consistency: This phenomenon has been observed in independent patient cohorts from different geographical locations (Netherlands and USA), suggesting a universal biological mechanism rather than a region-specific finding .

Methodologically, researchers investigating this phenomenon should employ quantitative ELISA assays to measure serum anti-RPL7/L12 IgG levels and include appropriate controls to rule out general bacterial exposure effects.

How can RPL7 antibodies be used in identifying ribosomal protein interactions?

RPL7 antibodies are valuable tools for investigating protein-protein interactions within the ribosomal complex. The protein interacts with several other ribosomal components, including RPL13 and RPL10A, and these interactions are essential for normal ribosomal function and protein synthesis .

To study these interactions, researchers can employ:

• Co-immunoprecipitation (Co-IP): Using RPL7 antibodies to pull down RPL7 and its interacting partners from cell lysates. This approach can identify both stable and transient interactions within the ribosomal complex.

• Proximity labeling methods: Combining RPL7 antibodies with techniques like BioID or APEX to identify proteins in close proximity to RPL7 within the cellular environment.

• Immunofluorescence co-localization: Using RPL7 antibodies alongside antibodies against potential interacting partners to visualize co-localization in cellular compartments.

When designing these experiments, researchers should consider:

• Using mild lysis conditions to preserve native protein complexes

• Including appropriate controls (IgG control, lysate input control)

• Validating interactions through multiple methodologies

• Considering the dynamic nature of ribosome assembly and function

The data from such studies can provide insights into both the structural organization of ribosomes and the regulatory networks that control protein synthesis in different cellular contexts.

What controls should be included when using RPL7 antibodies in research applications?

When designing experiments using RPL7 antibodies, researchers should implement a comprehensive set of controls to ensure data reliability and interpretability:

For Western Blotting:

• Positive control: Lysate from cells known to express RPL7 (ubiquitous in most cell types)

• Negative control: Lysate from cells treated with RPL7-specific siRNA, which should show significant reduction in signal intensity

• Loading control: Detection of a housekeeping protein (e.g., GAPDH, β-actin) to normalize protein loading

• Molecular weight marker: To confirm the detected band corresponds to the expected size of RPL7 (approximately 30 kDa)

For Immunoprecipitation:

• Input control: Small fraction of pre-IP lysate to confirm target protein presence

• IgG control: Parallel IP using isotype-matched non-specific IgG to identify non-specific binding

• Unrelated antibody control: IP with antibody against an unrelated protein to control for non-specific binding

• Reciprocal IP: If studying interactions, confirm by IP with antibodies against the putative interacting partner

For Immunohistochemistry/Immunofluorescence:

• Primary antibody omission: To detect non-specific binding of secondary antibody

• Blocking peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific staining

• Tissue from knockdown models: If available, tissues with reduced RPL7 expression should show diminished staining

Additional validation for ribosome-associated studies:

• RNase treatment controls: To distinguish RNA-dependent vs. protein-protein interactions

• Non-specific tag controls: When using tagged-ribosome approaches, control with non-specific epitope tags (e.g., Myc vs. HA)

What are the optimal conditions for using RPL7 antibodies in Western blotting?

Achieving optimal results with RPL7 antibodies in Western blotting requires careful attention to protocol details. Based on the available data, the following conditions have been validated for successful detection of RPL7:

Sample Preparation:

• Total cell lysates are generally sufficient as RPL7 is abundantly expressed in most cell types

• Protein extraction should be performed using buffers that preserve ribosomal integrity (e.g., RIPA buffer with protease inhibitors)

• Typical loading amounts range from 10-20 μg of total protein per lane

Blocking Conditions:

• 2% milk in an appropriate buffer (PBS or TBS) for 1 hour at 18°C has been demonstrated to be effective

• Alternative blocking agents like BSA may be used but should be validated

Primary Antibody Incubation:

• Dilution ratios typically range from 1:1000 to 1:2000 depending on the specific antibody

• Incubation in 2% milk solution for 18 hours at 4°C provides optimal results

• Shorter incubation times (e.g., 1-2 hours at room temperature) may be sufficient but should be validated

Detection System:

• HRP-conjugated secondary antibodies with appropriate species specificity (anti-rabbit for most commercially available RPL7 antibodies)

• Enhanced chemiluminescence (ECL) detection systems are commonly used

• Fluorescent secondary antibodies may offer advantages for quantitative analysis

Special Considerations:

• RPL7 has a molecular weight of approximately 30 kDa

• Due to post-translational modifications or proteolytic processing, additional bands may be observed

• siRNA knockdown controls are particularly valuable for confirming band specificity

What approaches should be used to validate RPL7 antibodies for novel applications?

Validating RPL7 antibodies for novel applications requires a systematic approach to ensure specificity and reliability. Researchers should implement the following validation strategies:

• Genetic Validation:

  • siRNA or shRNA knockdown: Demonstrate reduction in signal corresponding to RPL7 depletion

  • CRISPR-Cas9 knockout: Where feasible, complete elimination of the target should abolish specific signal

  • Overexpression systems: Detection of increased signal in cells overexpressing RPL7

• Analytical Validation:

  • Western blot analysis: Confirm single band of appropriate molecular weight

  • Mass spectrometry: Identify RPL7 in immunoprecipitated samples

  • Peptide competition: Pre-incubation with immunizing peptide should block specific binding

• Cross-Application Validation:

  • If validated for Western blot, test performance in IP and vice versa

  • Compare results across multiple detection methods for consistency

• Cross-Antibody Validation:

  • Use multiple antibodies targeting different epitopes of RPL7

  • Compare results between polyclonal and monoclonal antibodies

• Application-Specific Considerations:

  • For immunohistochemistry: Include tissue-specific positive and negative controls

  • For immunoprecipitation: Compare efficiency to established protocols

  • For proximity labeling: Validate with known ribosomal protein interactions

• Reproducibility Assessment:

  • Test across multiple cell lines or tissue types

  • Evaluate batch-to-batch consistency of antibody performance

  • Implement standardized protocols to ensure reproducibility

A comprehensive validation strategy increases confidence in antibody specificity and enables reliable interpretation of experimental results in novel applications.

How should researchers interpret contradictory results when using RPL7 antibodies?

When faced with contradictory results using RPL7 antibodies, researchers should implement a systematic approach to troubleshooting and interpretation:

In scientific literature, contradictions in RPL7 studies might stem from its dual roles - as a structural ribosomal component and as a regulatory factor in translation . When interpreting such contradictions, researchers should consider the specific cellular context and experimental conditions under which the observations were made.

What are common pitfalls in RPL7 antibody-based immunoprecipitation experiments?

Immunoprecipitation using RPL7 antibodies presents several challenges that researchers should anticipate and address:

• Non-specific Binding:

  • Ribosomal proteins are abundant and can bind non-specifically to beads or IgG

  • Solution: Include stringent washing steps and appropriate IgG controls

  • Validation: Compare results with non-specific antibody (e.g., anti-Myc) immunoprecipitation

• RNA-Dependent Interactions:

  • Some ribosomal protein associations are mediated by RNA rather than direct protein-protein interactions

  • Solution: Include parallel samples with RNase treatment to distinguish RNA-dependent interactions

  • Consideration: RPL7 binds to G-rich structures in RNA, which may influence interaction patterns

• Incomplete Ribosome Precipitation:

  • Standard IP protocols typically achieve around 25% efficiency in precipitating ribosomal complexes

  • Solution: Optimize lysis conditions and antibody-to-sample ratios

  • Quantification: Western blot for multiple ribosomal proteins to assess complex integrity

• Buffer Compatibility Issues:

  • Ribosomal integrity is sensitive to buffer conditions

  • Solution: Use buffers that maintain ribosomal structure while allowing effective antibody binding

  • Optimization: Test multiple buffer compositions with varying salt and detergent concentrations

• Contamination with Maternal Tissue:

  • In studies involving products of conception, maternal tissue contamination can complicate interpretation

  • Solution: Include microsatellite analysis when studying fetal tissues to differentiate maternal contamination

• Antibody Cross-Reactivity:

  • Some RPL7 antibodies may cross-react with RPL7A or bacterial RPL7/L12

  • Solution: Validate specificity with recombinant proteins or peptide competition assays

  • Consideration: Particularly important when studying bacterial-host interactions

By anticipating these challenges, researchers can design experiments with appropriate controls and optimization steps to ensure reliable results from RPL7 antibody-based immunoprecipitation studies.

How can researchers distinguish between normal RPL7 function and disease-associated alterations?

Distinguishing between normal RPL7 function and disease-associated alterations requires careful experimental design and interpretation:

By implementing these approaches, researchers can differentiate between normal biological variation in RPL7 function and genuine disease-associated alterations, leading to more accurate interpretation of experimental results in both basic research and clinical studies.