RPL36AL Antibody

What is RPL36AL and why is it studied in research?

RPL36AL (ribosomal protein L36a-like) is a component of the 60S subunit of cytoplasmic ribosomes. It belongs to the L44E (L36AE) family of ribosomal proteins and shares sequence similarity with yeast ribosomal protein L44. Cytoplasmic ribosomes, which catalyze protein synthesis, consist of a small 40S subunit and a large 60S subunit, together composed of 4 RNA species and approximately 80 structurally distinct proteins . RPL36AL is distinct from but nearly identical to ribosomal protein L36a (RPL36A), despite their similar names and protein products . As with many ribosomal proteins, RPL36AL has multiple processed pseudogenes dispersed throughout the genome . Research into RPL36AL often focuses on understanding ribosome assembly, function, and the role of ribosomal proteins in cellular homeostasis and disease states.

The distinction between polyclonal and monoclonal RPL36AL antibodies has significant implications for experimental design and interpretation:

Polyclonal RPL36AL antibodies:

• Generated in various hosts (typically rabbits) against multiple epitopes of the RPL36AL protein

• Recognize different regions of the target protein, potentially increasing detection sensitivity

• May show batch-to-batch variability in specificity and affinity

• Often used in applications like IHC where signal amplification is beneficial

• Example: The RPL36AL polyclonal antibody described in search result is generated against a recombinant fusion protein of human RPL36AL (NP_000992.1)

Monoclonal RPL36AL antibodies:

• Recognize a single epitope of the target protein

• Offer higher specificity and reproducibility between experiments

• May provide lower background but potentially less sensitivity than polyclonals

• Particularly valuable in applications requiring high specificity

For optimal experimental outcomes, researchers should select the appropriate antibody type based on the specific research question, required sensitivity, and application context.

What are the optimal protocols for Western blot analysis using RPL36AL antibodies?

For successful Western blot analysis of RPL36AL, researchers should follow these optimized protocols based on validated methods:

• Sample preparation:

  • Prepare cell or tissue lysates using standard lysis buffers containing protease inhibitors

  • Use 25μg protein per lane for adequate detection

• Antibody incubation parameters:

  • Primary antibody: Use RPL36AL antibody at 1:1000 dilution

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

• Blocking conditions:

  • Block membranes with 3% nonfat dry milk in TBST buffer

• Detection method:

  • Use ECL Basic Kit for chemiluminescent detection

  • Optimal exposure time of approximately 90 seconds has been validated for cell line extracts

• Controls to include:

  • Positive control: Jurkat whole cell lysate has been validated for RPL36AL expression

  • Negative control: Secondary antibody only to assess background

The expected molecular weight for RPL36AL is approximately 12 kDa , and researchers should be attentive to this region when analyzing blots. When troubleshooting, consider that the small size of this protein may require optimization of gel percentage and transfer conditions for optimal resolution.

How should RPL36AL antibodies be properly stored and handled to maintain activity?

Proper storage and handling of RPL36AL antibodies are critical for maintaining their activity and ensuring experimental reproducibility. Based on manufacturer recommendations:

• Short-term storage (up to one month):

  • Store at 4°C

  • Keep in the original container protected from light

• Long-term storage:

  • Store at -20°C

  • Aliquot antibody solution to avoid repeated freeze-thaw cycles

  • Typical formulation includes PBS with 0.02% sodium azide and 50% glycerol, pH 7.2-7.3

• Thawing procedure:

  • Thaw aliquots at room temperature or 4°C

  • Briefly centrifuge to collect solution at the bottom of the tube

  • Mix gently to ensure homogeneity before use

• Handling precautions:

  • Avoid contamination of antibody solutions

  • Some preparations may contain 0.1% BSA, which should be considered for certain applications

  • Follow appropriate safety procedures when handling solutions containing sodium azide

Following these storage and handling guidelines will help maintain antibody activity and specificity, leading to more consistent and reliable experimental results.

What are the recommended protocols for immunofluorescence using RPL36AL antibodies?

For optimal immunofluorescence detection of RPL36AL, researchers should follow these validated protocols:

• Cell preparation and fixation:

  • Culture cells (e.g., C6 cells) on glass coverslips

  • Fix with 4% paraformaldehyde and permeabilize with appropriate detergent

  • Block with normal serum matching the species of the secondary antibody

• Antibody incubation parameters:

  • Primary antibody: RPL36AL polyclonal antibody at a dilution of 1:100

  • Secondary antibody: Fluorophore-conjugated anti-species antibody at manufacturer's recommended dilution

  • Include DAPI counterstain for nuclear visualization

• Microscopy considerations:

  • Examine subcellular localization, expected primarily in the cytoplasm where ribosomes are abundant

  • Capture images at appropriate magnification to resolve ribosomal distribution

  • Use appropriate filter sets for the selected fluorophores

• Controls to include:

  • Primary antibody omission control to assess background and non-specific binding

  • Positive control tissues or cells with known RPL36AL expression

Expected results include cytoplasmic staining patterns consistent with ribosomal distribution, potentially with some nuclear signal in cells with active ribosome biogenesis. Results should be analyzed in conjunction with DAPI nuclear staining to assess subcellular localization accurately .

How can cross-reactivity between RPL36AL and RPL36A be addressed in experiments?

Addressing cross-reactivity between RPL36AL and RPL36A presents a significant challenge for researchers due to the high sequence similarity between these proteins. To ensure specificity:

• Antibody selection strategies:

  • Choose antibodies raised against unique regions or epitopes of RPL36AL

  • Review the immunogen information provided by manufacturers (e.g., recombinant fusion protein of human RPL36AL, NP_000992.1)

  • Request information about cross-reactivity testing from manufacturers

• Experimental validation approaches:

  • Conduct preliminary experiments with positive and negative controls

  • Perform peptide competition assays with specific RPL36AL and RPL36A peptides

  • Consider siRNA knockdown of RPL36AL to confirm antibody specificity

• Data analysis considerations:

  • Be aware that these are distinct genes despite encoding nearly identical proteins

  • Interpret results cautiously, acknowledging potential cross-reactivity

  • When possible, complement antibody-based detection with nucleic acid-based methods that can distinguish between the two genes

• Alternative strategies:

  • For gene expression studies, design PCR primers or probes unique to each gene

  • Consider using epitope-tagged versions of the proteins in overexpression studies

What are the considerations for using RPL36AL antibodies in disease-related research?

When incorporating RPL36AL antibodies into disease-related research, particularly cancer studies, researchers should consider several important factors:

• Disease associations and contexts:

  • Recent literature suggests potential associations between RPL36AL and cancer phenotypes

  • Evidence indicates that changes in receptor cell phenotypes associated with cancer may involve ribosomal protein signaling

• Tissue-specific expression considerations:

  • RPL36AL antibodies have been validated in various human tissues including:

    • Adult placenta

    • Fetal brain, heart, and lung

    • Breast cancer and liver cancer tissues

  • Expression patterns may vary between normal and disease states

• Experimental design recommendations:

  • Include appropriate diseased and normal tissue controls

  • Consider multiple detection methods (IHC, WB, IF) for comprehensive analysis

  • Use standardized scoring systems when conducting IHC studies in tissue samples

• Interpretation challenges:

  • Distinguish between alterations in ribosomal protein expression as drivers versus consequences of disease

  • Consider the broader context of ribosome biogenesis and translation regulation in disease mechanisms

  • Recognize that mutations in ribosomal proteins may have disease-specific effects beyond their canonical roles

Researchers investigating disease associations should be aware that ribosomal proteins like RPL36AL may have extraribosomal functions that contribute to pathogenesis, making careful experimental design and interpretation essential.

What controls should be included when using RPL36AL antibodies in research?

A robust control strategy is essential for reliable interpretation of experiments using RPL36AL antibodies:

• Primary controls for antibody specificity:

  • Positive control: Validated cell lines or tissues known to express RPL36AL (e.g., Jurkat cells)

  • Negative control: Either tissues known not to express the target or primary antibody omission

  • Peptide competition control: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Application-specific controls:

  • Western blot: Include molecular weight markers and loading controls (e.g., GAPDH, β-actin)

  • IHC/IF: Include isotype controls and secondary-antibody-only controls

  • Multi-color IF: Include single-color controls to assess bleed-through

• Experimental validation controls:

  • siRNA knockdown of RPL36AL to confirm specificity of antibody signal

  • Overexpression of tagged RPL36AL to confirm detection capability

  • Parallel detection using alternative antibodies targeting different epitopes

• Cross-reactivity assessment:

  • Given the similarity between RPL36AL and RPL36A, controls to assess potential cross-reactivity

  • Comparison with RPL36A-specific antibodies when available

  • Genetic manipulation approaches to distinguish between the proteins

Implementing a comprehensive control strategy enhances the reliability and interpretability of experimental results while providing evidence for antibody specificity and performance.

How can researchers validate the specificity of RPL36AL antibodies for their experimental system?

Validating antibody specificity is crucial for generating reliable data. For RPL36AL antibodies, researchers should implement these validation strategies:

• Genetic validation approaches:

  • siRNA/shRNA knockdown of RPL36AL followed by Western blot or immunostaining

  • CRISPR-Cas9 knockout of RPL36AL gene (when feasible)

  • Comparison of signal in wild-type versus genetically modified samples

• Biochemical validation methods:

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

  • Peptide competition assays using the immunizing peptide

  • Western blot analysis to confirm detection at the expected molecular weight (12 kDa)

• Orthogonal detection methods:

  • Correlation of protein detection with mRNA expression levels

  • Use of multiple antibodies targeting different epitopes of RPL36AL

  • Comparison with tagged recombinant RPL36AL expression

• System-specific validation:

  • Verify detection in positive control samples relevant to the experimental system

  • Assess expression patterns across different tissues or cell types

  • Compare with published literature on expected expression patterns

• Address potential RPL36A cross-reactivity:

  • Compare detection patterns with RPL36A-specific reagents

  • Use genetic approaches to distinguish between the two proteins

  • Consider epitope mapping to identify antibodies with minimal cross-reactivity

Thorough validation not only ensures experimental reliability but also contributes to reproducibility and confidence in research findings involving RPL36AL.

What are the common technical challenges in RPL36AL antibody-based experiments and how can they be addressed?

Researchers frequently encounter technical challenges when working with RPL36AL antibodies. Here are common issues and their solutions:

• Specificity concerns:

  • Challenge: Cross-reactivity with RPL36A due to high sequence similarity

  • Solution: Use antibodies raised against unique regions; validate with genetic approaches; consider using recombinant tagged versions in overexpression studies

• Detection sensitivity limitations:

  • Challenge: Low endogenous expression in some cell types or tissues

  • Solution: Optimize protein extraction methods; increase antibody concentration; employ signal amplification techniques; extend exposure times for Western blots

• Background and non-specific binding:

  • Challenge: High background in immunostaining or Western blot applications

  • Solution: Optimize blocking conditions (try 3% nonfat dry milk in TBST as validated) ; increase washing steps; adjust antibody dilutions; consider alternative secondary antibodies

• Fixation and epitope accessibility issues:

  • Challenge: Epitope masking during fixation for immunohistochemistry

  • Solution: Test different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0 have been recommended) ; optimize fixation protocols; consider alternative fixatives

• Small protein detection challenges:

  • Challenge: Detecting the 12 kDa RPL36AL protein on Western blots

  • Solution: Use higher percentage gels (15-20%); optimize transfer conditions for small proteins; consider using specialized membrane types with smaller pore sizes

• Result interpretation complexities:

  • Challenge: Distinguishing between RPL36AL and RPL36A signals

  • Solution: Include appropriate controls; confirm with alternative methods; consider the biological context and known expression patterns

By anticipating these challenges and implementing appropriate technical solutions, researchers can improve the reliability and reproducibility of their RPL36AL antibody-based experiments.

How can RPL36AL antibodies contribute to studies of ribosome biogenesis and function?

RPL36AL antibodies offer valuable tools for investigating ribosome biology through several advanced applications:

• Ribosome assembly studies:

  • Monitor RPL36AL incorporation into pre-ribosomal particles during biogenesis

  • Track subcellular localization during ribosome maturation using immunofluorescence

  • Assess changes in RPL36AL association with ribosomal subunits under different cellular conditions

• Translational regulation analysis:

  • Examine RPL36AL protein levels in response to translation inhibitors or cellular stress

  • Study potential extraribosomal functions through co-immunoprecipitation with non-ribosomal partners

  • Investigate post-translational modifications of RPL36AL that might regulate ribosome function

• Differential ribosome composition assessment:

  • Compare RPL36AL incorporation into ribosomes across different tissues or developmental stages

  • Analyze potential specialized ribosomes with unique translational properties

  • Study RPL36AL exchange dynamics in mature ribosomes

• Disease-related ribosome dysfunction:

  • Investigate changes in RPL36AL levels or localization in ribosomopathies

  • Assess potential alterations in cancer cells, where ribosome biogenesis is often dysregulated

  • Study the impact of RPL36AL mutations or expression changes on global translation

These applications leverage RPL36AL antibodies to provide insights into fundamental aspects of ribosome biology and potential connections to disease mechanisms, particularly in cancer where ribosomal protein alterations may contribute to pathogenesis .

What considerations should researchers take when using RPL36AL antibodies in tissue-specific expression studies?

When investigating tissue-specific expression patterns of RPL36AL, researchers should consider several important factors:

These considerations will help researchers generate reliable and biologically meaningful data when studying tissue-specific expression patterns of RPL36AL.

How does RPL36AL research contribute to understanding cancer biology and potential therapeutic targets?

Recent findings suggest important connections between RPL36AL and cancer biology, offering potential insights for therapeutic development:

• Ribosomal proteins in cancer pathogenesis:

  • Alterations in ribosomal proteins, including RPL36AL, have been associated with cancer phenotypes

  • Studies suggest that intercellular communication involving ribosomal components can reprogram receptor cells and change their phenotype in cancer contexts

  • RPL36AL antibodies enable investigation of these processes through detection and localization studies

• Diagnostic and prognostic applications:

  • Expression pattern analysis in cancer tissues using validated antibodies

  • Potential correlation of RPL36AL expression levels with disease progression or treatment response

  • Comparison between normal and malignant tissues to identify cancer-specific alterations

• Mechanistic investigations:

  • Study of RPL36AL in specialized ribosomes that may preferentially translate oncogenic mRNAs

  • Investigation of potential extraribosomal functions in cancer cell signaling

  • Analysis of RPL36AL interactions with cancer-relevant pathways

• Therapeutic implications:

  • Identification of cancer-specific dependencies on RPL36AL function

  • Exploration of synthetic lethality approaches targeting cancer cells with altered ribosome composition

  • Development of strategies to disrupt cancer-specific translation programs

• Research applications of RPL36AL antibodies:

  • Screening cellular responses to potential therapeutics targeting ribosome biogenesis

  • Monitoring changes in RPL36AL expression or localization following treatment

  • Identifying cancer subtypes with distinct RPL36AL expression patterns

These research directions highlight how RPL36AL antibodies contribute to fundamental understanding of cancer biology while potentially informing therapeutic strategies targeting ribosome function or composition in malignant cells.