RPL23A Antibody, FITC conjugated

What is RPL23A and why is it important in research applications?

RPL23A (Ribosomal Protein L23A) is a component of the 60S large ribosomal subunit with a calculated molecular weight of approximately 18 kDa (observed range: 18-23 kDa). This protein plays critical roles in ribosome assembly and protein translation processes. RPL23A is of particular interest in research because it interacts with nuclear chaperone proteins such as Bcp1 and has been implicated in ribosome biogenesis pathways . The protein contains 156 amino acids and is encoded by gene ID 6147 according to NCBI database references . For researchers, RPL23A serves as an important marker for studies involving ribosome structure, function, and cellular localization of translation machinery.

What applications are suitable for FITC-conjugated RPL23A antibodies?

FITC-conjugated RPL23A antibodies are particularly suitable for fluorescence-based applications including:

• Immunofluorescence microscopy (IF): Typically used at dilutions of 1:20-1:200

• Flow cytometry: For quantitative analysis of RPL23A expression in cell populations

• Immunocytochemistry (ICC): Effective for cellular localization studies

• Fluorescence-based immunohistochemistry: For tissue section analysis

When designing experiments, researchers should note that FITC has an excitation maximum at ~495 nm and emission maximum at ~519 nm, making it compatible with standard FITC filter sets on fluorescence microscopes. Unlike unconjugated antibodies that require secondary detection, FITC-conjugated antibodies enable direct visualization, reducing background and potential cross-reactivity issues in multi-label experiments.

How should researchers optimize fixation and permeabilization for FITC-conjugated RPL23A antibody staining?

Optimal fixation and permeabilization methods for RPL23A detection require balancing epitope preservation with cellular penetration:

For optimal RPL23A detection, antigen retrieval may be required for certain sample types. Published protocols suggest using TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative . When working with FITC-conjugated antibodies, it's important to perform fixation in the dark or under minimal light exposure to prevent photobleaching of the fluorophore. Researchers should validate the optimal protocol for their specific cell type or tissue, as RPL23A accessibility may vary depending on the sample preparation method.

What controls should be included when using FITC-conjugated RPL23A antibodies?

Proper experimental controls are essential for validating RPL23A antibody specificity and distinguishing true signal from background:

• Negative controls:

  • Isotype control: FITC-conjugated IgG of the same isotype as the RPL23A antibody (IgG2a for monoclonal or IgG for polyclonal antibodies )

  • Secondary antibody-only control (for indirect detection methods)

  • Unstained sample (for autofluorescence assessment)

• Positive controls:

  • Validated cell lines known to express RPL23A (e.g., HeLa, HepG2)

  • Known positive tissues (e.g., human liver, heart, or colon tissues)

• Specificity controls:

  • Pre-absorption with recombinant RPL23A protein

  • RNA interference to knock down RPL23A expression

  • Comparison with alternative RPL23A antibody clones targeting different epitopes

For advanced applications, researchers should include titration controls to determine the optimal antibody concentration that maximizes signal-to-noise ratio. For FITC-conjugated antibodies specifically, a photobleaching control (fixed time point imaging of a control sample) helps assess signal stability during extended imaging sessions.

How can researchers validate the specificity of FITC-conjugated RPL23A antibodies?

Validation of antibody specificity is crucial for ensuring experimental reliability:

• Western blot validation: Even when using FITC-conjugated antibodies for fluorescence applications, unconjugated versions of the same clone should first be validated by Western blot. Look for a single band at the expected molecular weight of 18-23 kDa in positive control samples like HeLa or HepG2 cell lysates .

• Immunoprecipitation: Confirm that the antibody can specifically pull down RPL23A from cell lysates. Validated protocols show successful immunoprecipitation from HepG2 cells using 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate .

• Knockout/knockdown validation: Compare staining between wild-type cells and those with reduced RPL23A expression through siRNA or CRISPR techniques.

• Cross-validation with multiple antibodies: Compare staining patterns using antibodies targeting different epitopes of RPL23A, such as those binding amino acids 1-92 versus 59-156 .

• Mass spectrometry validation: After immunoprecipitation, confirm the identity of pulled-down proteins through mass spectrometry to verify that RPL23A is indeed the captured target.

Research indicates that bcp1 mutant cells show unstable Rpl23 levels , suggesting these could serve as useful negative controls in yeast models to further validate antibody specificity.

What considerations are important when multiplexing FITC-conjugated RPL23A antibodies with other fluorophores?

Successful multiplexing requires careful consideration of spectral properties and antibody compatibility:

• Spectral separation: Choose fluorophores with minimal spectral overlap with FITC (ex/em: 495/519 nm). Good companion choices include:

  • DAPI (nuclear counterstain, ex/em: 358/461 nm)

  • Texas Red or Cy3 (red channel, ex/em: ~596/615 nm or ~550/570 nm)

  • Far-red dyes like Cy5 (ex/em: ~650/670 nm)

• Antibody host species compatibility: When using multiple primary antibodies, select those raised in different host species to avoid cross-reactivity. For example, pair a mouse monoclonal FITC-conjugated RPL23A antibody with rabbit antibodies against other targets.

• Bleed-through prevention:

  • Use sequential scanning on confocal microscopes

  • Include single-color controls for compensation setting

  • Consider linear unmixing algorithms for closely overlapping signals

• Co-localization studies: RPL23A interacts directly with Bcp1 chaperone protein , making this an interesting co-staining target. When designing such experiments, consider that interaction primarily occurs with free Rpl23 rather than ribosome-incorporated Rpl23 , which may influence staining patterns and interpretation.

How does the FITC conjugation affect antibody performance compared to unconjugated versions?

FITC conjugation introduces important differences that researchers should consider:

• Binding affinity: Conjugation can potentially alter epitope recognition, typically requiring titer optimization. While unconjugated RPL23A antibodies for Western blot may work at dilutions of 1:2000-1:12000 , FITC-conjugated versions often require more concentrated applications (typically 1:20-1:200 for IF).

• Steric hindrance: The addition of FITC molecules may affect antibody access to certain epitopes, particularly in densely packed cellular structures like ribosomes. This is especially relevant for RPL23A detection since this protein has specific interactions with nuclear chaperones that could be sterically disrupted .

• Signal stability: FITC is more prone to photobleaching than newer fluorophores. When imaging:

  • Minimize exposure times

  • Use anti-fade mounting media

  • Consider using oxygen scavengers in live-cell applications

• pH sensitivity: FITC fluorescence is optimal at slightly alkaline pH (7.5-8.5) and decreases in acidic environments. This is particularly relevant when imaging RPL23A in acidic cellular compartments.

• Storage requirements: FITC-conjugated antibodies typically require storage at -20°C in the dark, with 50% glycerol as a cryoprotectant . Aliquoting is recommended to prevent repeated freeze-thaw cycles that can degrade both antibody function and fluorophore activity.

How can researchers troubleshoot weak or inconsistent signals when using FITC-conjugated RPL23A antibodies?

When encountering signal issues, consider these methodological solutions:

For fixation-resistant samples, research indicates that RPL23A may require specialized extraction protocols. Studies have shown that certain cellular pools of RPL23A may be difficult to detect, particularly when the protein is bound to chaperones like Bcp1 . In such cases, try different fixation/extraction combinations or consider using antibodies targeting different epitopes, such as the N-terminal (AA 1-92) versus mid-protein regions (AA 59-156) .

What is the recommended protocol for quantifying RPL23A expression using FITC-conjugated antibodies?

For accurate quantification of RPL23A expression levels:

• Flow cytometry quantification:

  • Use standardized beads with known fluorophore molecules to create a calibration curve

  • Compare median fluorescence intensity (MFI) between samples

  • Include isotype controls to determine background thresholds

  • Consider using mean equivalent soluble fluorochrome (MESF) units for standardized reporting

• Immunofluorescence microscopy quantification:

  • Acquire images using consistent exposure settings

  • Measure mean fluorescence intensity in defined cellular compartments (cytoplasm, nucleus)

  • Use software like ImageJ/FIJI with consistent thresholding parameters

  • Normalize to cell number or area

• Relative quantification methods:

  • Compare RPL23A levels to housekeeping proteins

  • Use nuclear counterstains (DAPI) for normalization of cell number

  • Consider ratiometric imaging with a reference fluorophore

Research has demonstrated that free versus ribosome-incorporated RPL23A pools can be separated by ultracentrifugation (385,900 × g for 60 min) , which may be useful for quantifying different cellular pools of the protein in biochemical assays complementary to imaging studies.

How should researchers design optimal co-immunoprecipitation experiments with RPL23A?

When designing co-immunoprecipitation (co-IP) experiments to study RPL23A interactions:

• Lysis conditions optimization:

  • Use buffers that preserve protein-protein interactions (e.g., NP-40 or CHAPS-based buffers)

  • Include protease inhibitors to prevent degradation

  • Consider RNase treatment to distinguish RNA-dependent from direct protein interactions

• Antibody selection:

  • For IP applications, use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate

  • Consider using tag-based systems (e.g., HA, FLAG) for cleaner pull-downs

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Detection strategies:

  • For FITC-conjugated antibodies in IP-western experiments, consider how the fluorophore might impact SDS-PAGE migration

  • Include input, flow-through, and IP fractions as controls

  • Consider differential centrifugation to separate free from ribosome-bound RPL23A

Research has demonstrated that RPL23A interactions with partners like Bcp1 can be successfully detected through co-IP approaches . When seeking novel interaction partners, consider that RPL23A exists in both free and ribosome-incorporated forms, with different protein interaction profiles in each state. Experiments separating these pools (such as with ultracentrifugation at 385,900 × g for 60 min) may reveal state-specific interactions .

How can FITC-conjugated RPL23A antibodies be used to study ribosome biogenesis defects?

FITC-conjugated RPL23A antibodies offer powerful tools for investigating ribosome biogenesis:

• Nucleolar stress response:

  • Monitor nucleolar morphology changes under stress conditions

  • Quantify RPL23A redistribution between nucleolus, nucleoplasm, and cytoplasm

  • Compare with other nucleolar markers to assess specificity of response

• Genetic perturbation analysis:

  • In yeast models, bcp1 mutations lead to RPL23A instability

  • Human cells with ribosome biogenesis defects can show altered RPL23A localization

  • Measure RPL23A levels before and after genetic interventions

• Drug response studies:

  • Assess how ribosome biogenesis inhibitors affect RPL23A expression and localization

  • Monitor recovery kinetics after drug withdrawal

  • Compare with rRNA synthesis markers for comprehensive analysis

When designing such experiments, consider that free RPL23A (not incorporated into ribosomes) can be stabilized by chaperone proteins like Bcp1 . This suggests that proper folding and stability of RPL23A might be rate-limiting steps in ribosome assembly under certain conditions, making it an informative marker for biogenesis defects.

What are the current limitations in RPL23A research that could be addressed with improved antibody technology?

Current research gaps that could benefit from advanced RPL23A antibody approaches include:

• Distinguishing free vs. ribosome-incorporated RPL23A:

  • Current antibodies typically detect total RPL23A pools

  • Conformation-specific antibodies could potentially distinguish between free and incorporated forms

  • This distinction is important as research shows these pools have different stability and interaction properties

• Temporal dynamics:

  • Photobleaching limits long-term imaging with FITC

  • Newer fluorophores or photoconvertible tags could enable better pulse-chase experiments

  • Understanding assembly kinetics requires stable fluorophores

• Structural biology interfaces:

  • Current antibodies may disrupt functional interactions

  • Smaller probes (nanobodies, aptamers) conjugated to FITC might provide less invasive labeling

  • Accessibility of epitopes within assembled ribosomes remains challenging

• Cross-species comparability:

  • Existing antibodies show variable cross-reactivity between human, mouse, and rat RPL23A

  • More consistent cross-species reactivity would facilitate comparative studies

  • Targeting highly conserved epitopes could improve cross-reactivity

• Quantitative accuracy:

  • Absolute quantification of RPL23A molecules remains challenging

  • Calibrated fluorescence standards could improve quantitative comparisons

  • Single-molecule sensitivity would enable detection of rare intermediates

Addressing these limitations could significantly advance our understanding of RPL23A's role in ribosome biogenesis and function across different biological systems.