RPL14 Antibody

What is RPL14 and what are its cellular functions?

RPL14 (Ribosomal Protein L14) is a component of the large 60S ribosomal subunit, belonging to the L14E family of ribosomal proteins. It contains a basic region-leucine zipper (bZIP)-like domain . As part of the ribosome, RPL14 plays a critical role in the ribonucleoprotein complex responsible for protein synthesis in cells . Beyond its canonical ribosomal function, RPL14 has been reported to bind specifically to the 3'-terminal U-tract of U6 snRNA, suggesting potential involvement in pre-mRNA splicing . Recent research has also identified potential extra-ribosomal functions, including tumor suppression in certain cancers .

The species reactivity of RPL14 antibodies varies by product:

When working with non-validated species, preliminary testing is strongly recommended due to potential variability in cross-reactivity despite sequence homology.

What is the expected molecular weight of RPL14 in Western blot applications?

While the calculated molecular weight of RPL14 is approximately 23 kDa , the observed molecular weight in Western blot can vary:

This variation may be attributed to post-translational modifications, sample preparation methods, or differences in gel systems. The RPL14 protein contains a polymorphic trinucleotide (GCT) repeat tract resulting in a variable stretch of alanine residues, which may contribute to migration differences .

What are the recommended storage conditions for RPL14 antibodies?

Proper storage is critical for maintaining antibody activity and specificity:

Always centrifuge briefly prior to opening vials to collect all material at the bottom of the tube .

How should I optimize Western blot protocols for reliable RPL14 detection?

Western blot optimization for RPL14 requires careful attention to several parameters:

Sample Preparation:

• Use fresh lysates from validated positive control cell lines (HeLa, 293T, Jurkat, MCF-7)

• Load appropriate amounts of protein (typically 20 μg per lane)

• Include protease inhibitors in lysis buffers to prevent degradation

Antibody Parameters:

• Primary antibody dilution: Start with manufacturer's recommendation, then optimize (commonly 1:2000-1:50000)

• Secondary antibody: Goat Anti-Rabbit IgG, Peroxidase conjugated at 1:10000 dilution

• Blocking: 5% non-fat dry milk in TBST is effective for most RPL14 antibodies

Detection Conditions:

• Validated exposure times vary by cell line and antibody concentration

• Expected band patterns:

  • Single band at ~23-30 kDa in most cell lines

  • Multiple bands may indicate degradation or isoforms

As shown in validation data from Boster Bio, RPL14 antibody (M07781) at 1:2000 dilution successfully detected bands in multiple cell lines including HeLa, Jurkat, MCF-7, 293T, U-2OS, and A431 .

What are the key differences between polyclonal, monoclonal, and recombinant RPL14 antibodies?

Understanding antibody type differences is critical for experimental design:

For RPL14 research requiring high reproducibility between experiments, recombinant monoclonal antibodies like Abcam's EPR12301 offer advantages of consistency and specificity . For detection of conformationally altered RPL14 or in applications where sensitivity is paramount, polyclonal options may be preferable.

How can I validate the specificity of RPL14 antibodies for my experimental system?

Rigorous antibody validation is essential for reliable research outcomes:

Validation Approaches for RPL14 Antibodies:

• Positive Controls:

  • Test on cell lines with known RPL14 expression (HeLa, Jurkat, MCF-7, HepG2)

  • Comparison with recombinant RPL14 protein standards

• Genetic Controls:

  • siRNA/shRNA knockdown (complete knockout may be lethal due to essential ribosomal function)

  • Overexpression systems with tagged RPL14

• Method-Specific Validation:

  • WB: Verify single band of expected molecular weight (23-30 kDa)

  • IF: Confirm predominantly cytoplasmic localization with nucleolar enrichment

  • IHC: Compare with known expression patterns and include tissue negative controls

• Multi-antibody Approach:

  • Test multiple antibodies targeting different RPL14 epitopes

  • Compare results between polyclonal and monoclonal antibodies

• Blocking Peptide Competition:

  • Pre-incubate antibody with immunizing peptide to confirm signal specificity

Boster Bio's validation demonstrates RPL14 antibody specificity by showing consistent cytoplasmic staining in U-2OS cells by immunofluorescence and specific bands at the expected molecular weight across multiple cell lines by Western blot .

What are the considerations for using RPL14 antibodies in cancer research?

Recent research has implicated RPL14 in cancer biology, particularly as a potential tumor suppressor:

Key Research Findings:

• RPL14 expression is significantly reduced in nasopharyngeal carcinoma (NPC) tissues and cells

• RPL14 protein expression correlates with NPC clinical pathological features, including T and N classification of TNM staging (p < 0.05)

• RPL14 overexpression represses NPC cell proliferation, migration, and invasion

• RPL14 overexpression blocks NPC cells in S phase of the cell cycle

• RPL14 expression correlates with epithelial-mesenchymal transition (EMT) biomarkers including E-cadherin, N-cadherin, and vimentin

Methodological Considerations:

• Sample Selection:

  • Include matched tumor and adjacent normal tissues

  • Consider patient clinical parameters (stage, grade, treatment history)

• Expression Analysis:

  • Use multiple methods (IHC, WB, qRT-PCR) for comprehensive assessment

  • Quantify expression levels using proper normalization

• Functional Studies:

  • Pair antibody-based detection with functional assays (proliferation, migration, invasion)

  • Correlate RPL14 levels with cancer hallmark phenotypes

• Controls and References:

  • Include known RPL14-expressing and non-expressing cell lines

  • Compare with other ribosomal proteins to distinguish RPL14-specific effects

Research by demonstrates that RPL14 may function as an antioncogene in NPC, suggesting broader implications for understanding RPL14's role in other cancers.

How do I troubleshoot weak or non-specific signals when using RPL14 antibodies?

Common issues and solutions when working with RPL14 antibodies:

Method-Specific Troubleshooting:

Western Blot:

• For weak signals, Proteintech recommends concentrating the primary antibody (14991-1-AP) to 1:5000 instead of 1:50000

• Non-specific bands can be reduced by extending the blocking time to 1-2 hours at room temperature

• For RPL14 detection, 5% NFDM/TBST has been validated as an effective blocking/dilution buffer

Immunofluorescence:

• Fixation with 4% paraformaldehyde followed by 0.1% Triton X-100 permeabilization provides optimal results for RPL14 detection

• Specific signal enhancement can be achieved by using Dylight®488-conjugated secondary antibodies at 1:200 dilution

Immunohistochemistry:

• Antigen retrieval with EDTA buffer pH 9.0 has been validated for RPL14 detection in paraffin sections

• Alternative methods using citrate buffer pH 6.0 may be tested if initial results are unsatisfactory

How can RPL14 antibodies be used to investigate ribosome biogenesis and function?

RPL14 antibodies provide valuable tools for studying ribosome biology:

Research Applications:

• Ribosome Assembly Studies:

  • Immunoprecipitation to isolate RPL14-containing ribosomal complexes

  • Co-IP to identify RPL14 interaction partners during assembly

  • Western blotting to monitor RPL14 incorporation into 60S subunits

• Cellular Stress Responses:

  • Track RPL14 levels and localization during various cellular stresses

  • Correlate with nucleolar stress markers

  • Monitor RPL14 in response to translation inhibitors

• Ribosome Heterogeneity Analysis:

  • Examine RPL14 incorporation across different ribosome populations

  • Study specialized ribosomes in different tissues or disease states

  • Investigate post-translational modifications of RPL14

• Developmental Biology:

  • Track RPL14 expression during embryonic development

  • Examine tissue-specific ribosome composition

Methodological Approaches:

• Polysome Profiling: Use RPL14 antibodies to identify RPL14-containing fractions

• SILAC/Mass Spectrometry: Combine with RPL14 IP for quantitative proteomic analysis

• Proximity Labeling: Use RPL14 antibodies to verify BioID or APEX2 proximity labeling results

• Super-Resolution Microscopy: Visualize RPL14 within ribosomal structures at nanoscale resolution

RPL14's confirmed binding to the 3'-terminal U-tract of U6 snRNA suggests additional roles in pre-mRNA splicing , making it valuable for studying connections between ribosome biology and RNA processing.

What is the significance of RPL14 expression in pathological conditions?

Recent evidence suggests RPL14 has roles beyond ribosomal function:

Cancer Biology:

• RPL14 shows significantly reduced expression in nasopharyngeal carcinoma (NPC) tissues and cells

• RPL14 protein expression is associated with NPC clinical pathological features, including T and N classification of TNM staging

• RPL14 overexpression represses NPC cell proliferation, blocking cells in S phase

• RPL14 suppresses cell migration and invasion in NPC

• RPL14 correlates with epithelial–mesenchymal transition (EMT) biomarkers (E-cadherin, N-cadherin, vimentin)

These findings suggest RPL14 may function as an antioncogene in NPC and potentially in other cancers, offering new research directions for understanding the extra-ribosomal functions of ribosomal proteins in disease pathogenesis.

How do I select appropriate controls for RPL14 antibody experiments?

Proper experimental controls are essential for generating reliable data:

Positive Controls:

• Cell lines: HeLa, 293T, Jurkat, MCF-7, HepG2, and U-2OS cells show consistent RPL14 expression

• Tissues: Heart muscle tissue has been validated for RPL14 IHC detection

• Recombinant proteins: Consider using RPL14 recombinant proteins as standards for quantification

Negative Controls:

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

• Isotype controls: Rabbit IgG at equivalent concentration can control for non-specific binding

• Blocking peptide competition: Pre-incubation with immunizing peptide should eliminate specific signal

Method-Specific Controls:

When designing experiments, include biological replicates and technical controls to account for natural variation and experimental artifacts.

What are the detailed protocols for using RPL14 antibodies in different applications?

Western Blot Protocol for RPL14 Detection:

• Sample Preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Quantify protein concentration by BCA or Bradford assay

  • Prepare 20 μg protein per lane in Laemmli buffer with DTT

  • Heat samples at 95°C for 5 minutes

• Gel Electrophoresis and Transfer:

  • Separate proteins on 12% SDS-PAGE gel

  • Transfer to PVDF membrane (wet transfer recommended)

• Antibody Incubation:

  • Block membrane with 5% NFDM/TBST for 1 hour at room temperature

  • Incubate with primary RPL14 antibody at 1:2000-1:10000 dilution overnight at 4°C

  • Wash 3×10 minutes with TBST

  • Incubate with HRP-conjugated secondary antibody (1:10000) for 1 hour at room temperature

  • Wash 3×10 minutes with TBST

• Detection:

  • Apply ECL substrate and image

  • Expected band at 23-30 kDa depending on the specific antibody

Immunofluorescence Protocol:

• Sample Preparation:

  • Fix cells with 4% paraformaldehyde (10 min)

  • Permeabilize with 0.1% Triton X-100 (10 min)

• Staining:

  • Block with 2% BSA (1 hour at room temperature)

  • Incubate with RPL14 primary antibody (1:25-1:200) for 60 min at 37°C

  • Wash 3× with PBS

  • Incubate with fluorochrome-conjugated secondary antibody (1:200) for 40 min at 37°C

  • Counterstain with DAPI

  • Mount and image

Flow Cytometry Protocol:

• Sample Preparation:

  • Fix cells with 2% paraformaldehyde (10 min)

  • Permeabilize with 90% methanol (10 min)

  • Block with 2% BSA

• Antibody Staining:

  • Incubate with RPL14 antibody (1:25) for 60 min at 37°C

  • Wash with PBS

  • Incubate with fluorochrome-conjugated secondary antibody (1:200) for 40 min at 37°C

  • Analyze, acquiring >10,000 events per sample

These protocols are based on validated methods described in the product literature , but may require optimization for specific experimental conditions.

What are the epitope characteristics of different RPL14 antibodies?

Understanding the target epitope is crucial for antibody selection and experimental design:

The central region targeted by Boster Bio's antibody (amino acids 117-147) corresponds to a functionally important domain in RPL14. When selecting antibodies for specific applications, consider:

• Epitope accessibility in your application (native vs. denatured protein)

• Potential post-translational modifications near the epitope

• Conservation of the epitope sequence between species

• Potential overlap with protein interaction sites

For applications requiring detection of specific RPL14 variants or modified forms, select antibodies whose epitopes do not overlap with the regions of interest.