RPL12 Antibody

What is RPL12 and what cellular functions does it perform?

RPL12 (ribosomal protein L12) is a component of the large ribosomal subunit (60S) that plays a crucial role in protein synthesis. It binds directly to 26S ribosomal RNA and is part of the ribonucleoprotein complex responsible for cellular protein synthesis . Beyond its canonical role in translation, RPL12 has been implicated in DNA damage response pathways, particularly in UV-induced DNA damage repair .

When conducting research on RPL12, consider its dual functionality:

• Primary role in ribosomal structure and protein synthesis

• Secondary roles in cellular stress responses and DNA repair mechanisms

The protein has a calculated molecular weight of 18 kDa but typically migrates at approximately 21 kDa on SDS-PAGE gels , likely due to post-translational modifications.

Storage and handling recommendations vary by manufacturer and formulation:

For optimal stability:

• Avoid repeated freeze-thaw cycles by preparing appropriate aliquots

• Some formulations contain 0.02% sodium azide and 50% glycerol at pH 7.3

• Small volume sizes (20μl) may contain 0.1% BSA for additional stability

• Allow antibodies to equilibrate to room temperature before opening

How can I validate the specificity of an RPL12 antibody?

Rigorous antibody validation is essential for reliable results. Based on current methodologies , consider these approaches:

Multiple Validation Strategies:

• Peptide inhibition assays: Pre-incubate antibody with immunizing peptide before application to sample

• Knockdown experiments: Compare signal between control and RPL12 siRNA-treated cells (48h post-transfection optimal)

• Western blot profile analysis: Verify single band at expected molecular weight (~21 kDa)

• Positive control tissues/cells: Use validated samples (A549, Jurkat, HeLa cells for WB; human placenta/endometrial cancer tissue for IHC)

• Cross-species reactivity testing: Confirm signals in predicted reactive species (human, mouse, rat)

For phosphosite-specific antibodies, additional validation using phosphosite mutants may be required . When evaluating validation data, remember that reactivity may differ between applications (e.g., an antibody may work in WB but not IHC).

What is known about RPL12's role in UV-induced DNA damage response?

Recent research has identified non-canonical functions of RPL12 in DNA repair mechanisms:

• BmRPL12 (Bombyx mori RPL12) participates in UV-induced DNA damage repair pathways

• Knockdown experiments demonstrate its functional importance:

  • RPL12 siRNA transfection in BmN cells showed impaired DNA damage repair

  • Cells were exposed to 60 J/m² UVC after 48h of knockdown

  • DNA damage was quantified using CPD ELISA Kit at different time points

  • Statistical analysis using single-factor analysis of variance confirmed significance

Experimental approach for studying RPL12 in DNA damage:

• Conduct survival assays following UV exposure (0.15-0.3 J/m²)

• Use localization analysis with fluorescent fusion proteins (mCherry-tagged RPL12)

• Analyze truncated versions of RPL12 (amino acids 1-110 and 97-179) to determine functional domains

• Co-immunoprecipitation assays to identify interaction partners in response to UV damage

How do I troubleshoot weak or inconsistent signals when using RPL12 antibodies?

When encountering suboptimal results with RPL12 antibodies, consider these methodological adjustments:

Western Blot Troubleshooting:

• Sample preparation: Use RIPA lysis buffer with 10% PMSF for efficient extraction

• Loading control selection: α-Tubulin is recommended as loading control

• Secondary antibody optimization: Use species-specific HRP-conjugated antibodies at 1:1000 dilution

• Signal development: ECL technique with 3-minute exposure time has been validated

• Gel concentration: 12% SDS-PAGE provides optimal separation for RPL12

IHC Troubleshooting:

• Antigen retrieval optimization: Test both TE buffer pH 9.0 and citrate buffer pH 6.0

• Blocking optimization: Adjust blocking buffer composition (BSA percentage)

• Incubation time adjustment: Extend primary antibody incubation (overnight at 4°C)

• Detection system selection: Choose appropriate detection system based on tissue type

Always remember that "it is recommended that this reagent should be titrated in each testing system to obtain optimal results" as sample-dependent variations are common .

What controls should I include when designing experiments with RPL12 antibodies?

Robust experimental design requires appropriate controls:

Positive Controls:

• Cell lines: A549, Jurkat, and HeLa cells show consistent RPL12 expression

• Tissues: Human placenta, endometrial cancer, mouse/rat brain, and mouse/rat stomach tissues

Negative Controls:

• Primary antibody omission: Include samples treated identically but without primary antibody

• Isotype controls: Use matched isotype (IgG or IgG1) from same species (rabbit or mouse)

• Knockdown samples: RPL12 siRNA-treated cells (48h post-transfection)

Technical Validation Controls:

• Molecular weight marker: To confirm the 21 kDa observed band size

• Peptide competition: Pre-adsorption with immunizing peptide should abolish specific signal

• Cross-reactivity testing: Test antibody on multiple species if working with non-human models

How can I design co-immunoprecipitation experiments using RPL12 antibodies?

For studying RPL12 protein-protein interactions:

Co-IP Protocol Framework:

• Prepare cell lysates using RIPA lysis buffer containing 10% PMSF

• Incubate lysates with anti-RPL12 antibody (1:200-1:500 dilution) overnight at 4°C

• Add Protein A/G Sepharose beads and incubate for 2-4 hours

• Wash beads thoroughly to remove non-specific interactions

• Elute bound proteins with 5× SDS loading buffer

• Analyze by Western blotting using antibodies against suspected interaction partners

Experimental Variables to Consider:

• Compare normal conditions with stress conditions (e.g., 60 J/m² UVC treatment)

• Include reciprocal IP experiments (IP with partner protein antibody, detect RPL12)

• Use tagged constructs (FLAG-tag) for validation of interactions

• Include proper controls (IgG control, input samples, beads-only control)

This approach has successfully identified interaction between BmRPL12 and Bm65 in the context of UV-induced DNA damage response .

What explains the discrepancy between calculated (18 kDa) and observed (21 kDa) molecular weight of RPL12?

The difference between calculated (18 kDa) and observed (21 kDa) molecular weight is consistently reported across multiple sources and likely reflects:

• Post-translational modifications: Phosphorylation, acetylation, or other modifications can increase apparent molecular weight

• Protein structure factors: Hydrophobic residues or charged regions can affect SDS binding and migration

• Technical factors: Gel concentration and running conditions can influence migration patterns

When confirming RPL12 identity:

• Look for a band at approximately 21 kDa in Western blots

• Verify using multiple antibodies targeting different epitopes

• Consider analyzing post-translational modifications if the discrepancy is relevant to your research question

What are the immunogen characteristics of available RPL12 antibodies?

Understanding immunogen details helps predict epitope location and potential cross-reactivity:

When selecting an antibody:

• Choose epitopes relevant to your research question (e.g., N-terminal vs. C-terminal)

• Consider potential masking of epitopes by protein-protein interactions

• Evaluate cross-reactivity profiles across species based on epitope conservation

How should I optimize fixation and retrieval methods for RPL12 immunohistochemistry?

Successful IHC with RPL12 antibodies requires optimization of fixation and antigen retrieval:

Validated Protocols:

• Fixation: Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used

• Section thickness: 4-6 μm sections are optimal for most applications

• Primary antigen retrieval recommendation: TE buffer at pH 9.0

• Alternative retrieval method: Citrate buffer at pH 6.0

• Antibody dilution range: 1:100-1:2000 (optimization required for each tissue type)

• Incubation conditions: Typically overnight at 4°C or 1-2 hours at room temperature

Tissue-Specific Considerations:

• Human placenta, endometrial cancer tissue: Strong RPL12 expression

• Mouse/rat brain and stomach tissues: Consistent RPL12 detection

• Human gastric carcinoma: Validated for RPL12 immunostaining

• Mouse muscle tissue: Successful RPL12 detection reported

What approaches can I use to study RPL12 localization and truncation mutants?

To investigate RPL12 cellular localization and functional domains:

Experimental Approaches:

• Fluorescent fusion constructs:

  • Full-length RPL12-mCherry (amino acids 1-179)

  • N-terminal fragment (amino acids 1-110)

  • C-terminal fragment (amino acids 97-179)

• Visualization technique:

  • Transfect constructs into appropriate cell lines (BmN cells validated)

  • Fix with 4% paraformaldehyde after 48h

  • Permeabilize with 0.1% Triton X-100

  • Counterstain nuclei with DAPI

  • Image using fluorescence microscopy

• Functional analysis:

  • Test truncation mutants in complementation assays

  • Assess localization changes under stress conditions (UV exposure)

  • Combine with interactome studies to map domain-specific interactions

This approach has been successfully employed to determine the functional domains of RPL12 involved in UV damage response .

Beyond canonical functions, what emerging roles of RPL12 should researchers investigate?

Recent research has revealed non-canonical functions of RPL12 that warrant further investigation:

• DNA damage response: Role in UV-induced DNA damage repair pathways

• Transcriptional regulation: Potential association with RNA polymerase complexes

• RNA binding: Interaction with G-quadruplex structures suggested in publications

• Cell cycle regulation: Potential connection to CDK1-mediated proliferation control

• Mitochondrial functions: Association with mitochondrial RNA polymerase to activate transcription (mentioned in citations)

Research approaches to explore these functions:

• Comprehensive interactome analysis under various cellular stresses

• ChIP-seq to identify potential DNA binding sites

• RNA-binding protein immunoprecipitation (RIP) to characterize RNA interactions

• CRISPR-Cas9 knockout studies to delineate essential vs. non-essential functions

• Tissue-specific conditional knockouts to assess developmental roles

How can I design antibody specificity testing for RPL12 using peptide arrays?

Peptide arrays provide comprehensive specificity validation for antibodies:

Methodological Approach:

• Array design: Include RPL12 peptides spanning the full protein sequence in overlapping fragments

• Control peptides: Include related ribosomal proteins to assess cross-reactivity

• Modification-specific arrays: If studying post-translational modifications, include modified and unmodified peptide pairs

• Detection systems: Use fluorescently labeled secondary antibodies for quantitative analysis

• Data analysis: Calculate signal-to-noise ratios for each peptide spot

The AbSurance™ Antibody Specificity arrays mentioned in the search results provide a model for this approach . When designing a custom array:

• Include 15-20 amino acid peptides with 5-10 amino acid overlaps

• Incorporate known or predicted post-translational modification sites

• Include peptides from closely related proteins (other ribosomal proteins)

• Print peptides in multiple concentrations for dose-response assessment

This comprehensive approach provides detailed epitope mapping and cross-reactivity profiling beyond what basic Western blot validation can offer.

What are best practices for quantifying RPL12 protein expression changes?

For accurate quantification of RPL12 expression:

Western Blot Quantification:

• Use ImageJ software (version 1.54f or later) for band intensity quantification

• Include appropriate loading controls (α-Tubulin validated)

• Normalize RPL12 signal to loading control

• Use multiple biological replicates (minimum n=3)

• Apply statistical analysis (ANOVA recommended for multiple comparisons)

IHC Quantification:

• Use digital image analysis software for consistent scoring

• Establish scoring criteria (intensity, percentage of positive cells)

• Employ blinded assessment by multiple observers

• Include appropriate positive and negative controls in each batch

• Report both intensity and distribution of staining

Additional Quantitative Approaches:

• Mass spectrometry for absolute quantification

• ELISA for high-throughput screening

• Fluorescence-based quantification for subcellular localization studies