RPL26L1 Antibody

What is RPL26L1 and how does it differ from RPL26?

RPL26L1 (ribosomal protein L26-like 1) belongs to the universal ribosomal protein uL24 family and shares high sequence similarity with ribosomal protein L26. While RPL26 is a well-characterized component of the 60S ribosomal subunit, RPL26L1 is a paralog that differs by only two amino acids from RPL26 . Studies have shown that RPL26 is approximately 15 times more abundant than RPL26L1 in cells, though both are detected in ribosomal fractions . It remains unclear whether RPL26L1 functions primarily as a ribosomal protein or has evolved functions independent of the ribosome .

What are the key molecular characteristics of RPL26L1 that researchers should be aware of?

RPL26L1 has a calculated molecular weight of 17 kDa (145 amino acids), though it typically appears at 22-25 kDa on Western blots due to post-translational modifications . The protein is encoded by gene ID 51121 (NCBI) with UniProt ID Q9UNX3 . RPL26L1 plays a role in protein synthesis and has been associated with autism spectrum disorder . Researchers should note that RPL26L1 contains important lysine residues (K132 and K134) that are targets for UFMylation, a post-translational modification that appears to regulate its function .

How should researchers choose between antibodies that target only RPL26L1 versus those that detect both RPL26 and RPL26L1?

When selecting antibodies, researchers should consider:

• Research objective: If studying specific functions of RPL26L1 distinct from RPL26, choose antibodies raised against unique epitopes of RPL26L1 (often C-terminal regions) .

• Cross-reactivity concerns: Due to high sequence homology, many antibodies detect both proteins. Product 16487-1-AP specifically targets RPL26L1 in Western blot, IHC, and IF/ICC applications .

• Validation data: Examine if the antibody has been validated in your experimental system. For example, some antibodies have been tested in specific cell lines like HEK-293, K-562, and MCF-7 cells .

• Distinguishing paralogs: For distinguishing between the two paralogs, consider using antibodies targeting regions containing the differentiating amino acids, or using mass spectrometry approaches as described in published studies .

What are the optimal dilution ratios for different applications of RPL26L1 antibodies?

Based on validated protocols for RPL26L1 antibodies, researchers should consider the following dilution ranges:

It's important to titrate the antibody concentration in each specific experimental system to obtain optimal results, as detection sensitivity can be sample-dependent .

What antigen retrieval methods are recommended for immunohistochemistry with RPL26L1 antibodies?

For optimal results in IHC applications, researchers should perform heat-mediated antigen retrieval using either:

• TE buffer (pH 9.0) - primary recommended method for most tissues .

• Citrate buffer (pH 6.0) - alternative method that has been validated for certain tissue types including human stomach tissue .

Specific protocols have been validated for different tissue types. For example, RPL26L1 detection in mouse testis tissue requires formaldehyde fixation followed by heat-mediated antigen retrieval with citrate buffer . For human samples like brain tissue, antibodies such as A15759-1 have been validated using paraffin-embedded sections with antigen retrieval at 1:100 dilution (4°C) .

How can I validate the specificity of my RPL26L1 antibody and distinguish it from RPL26 detection?

To validate specificity:

• Use positive and negative controls: Test in cell lines with known expression levels (HEK-293, K-562, MCF-7 are positive controls for RPL26L1) .

• Peptide competition assay: Use blocking peptides derived from the specific immunogen to confirm specificity .

• Knockdown/knockout validation: Compare detection in wild-type versus RPL26L1-depleted samples.

• Parallel detection: Use antibodies targeting unique epitopes of RPL26 and RPL26L1 and compare migration patterns.

• Mass spectrometry validation: For definitive identification, use techniques that can distinguish the two paralogous proteins as described in research that identified distinguishing peptides .

What is known about the UFMylation of RPL26L1 and its biological significance?

RPL26L1, like RPL26, undergoes post-translational modification by UFM1 (UFMylation). Research indicates that:

• Modification sites: UFMylation primarily occurs at lysine residues K132 and K134 in the C-terminal region of RPL26L1 .

• Detection pattern: UFMylated RPL26L1 appears as two distinct species in Western blots (S1 and S2), corresponding to mono- and di-UFMylated forms .

• Functional significance: UFMylation of RPL26 and likely RPL26L1 is involved in translocation-associated quality control, particularly for proteins targeted to the endoplasmic reticulum (ER) .

• Degradation regulation: RPL26 UFMylation facilitates the degradation of certain ER-targeted polypeptides during translational arrest, a function that may also apply to RPL26L1 .

• Ribosomal context: The modified lysine residues are solvent-exposed and proximal to the polypeptide exit tunnel, suggesting functional relevance for co-translational processes .

Recent studies have identified RPL26(L1) as the principal target of UFMylation in HEK293 cells, highlighting its importance in this cellular pathway .

How is RPL26L1 integrated into ribosomes, and does it have functions independent of the ribosome?

The ribosomal integration of RPL26L1 appears complex:

• Ribosomal incorporation: Sucrose cushion purification experiments confirm that RPL26L1 is incorporated into ribosomes .

• Structural position: As a member of the uL24 family, RPL26L1 likely occupies a position near the polypeptide exit tunnel in the large ribosomal subunit .

• Potential independent functions: While RefSeq notes that "it is not currently known whether the encoded protein is a functional ribosomal protein or whether it has evolved a function that is independent of the ribosome" , its detection in ribosomal fractions supports a ribosomal role .

• Modification requirements: Importantly, preventing RPL26 incorporation into ribosomes (by FLAG tagging at its amino terminus, which is buried in ribosomes) abrogated UFMylation, suggesting modification occurs in the ribosomal context .

Research suggests RPL26L1 may have specialized functions related to translation quality control, particularly for ER-targeted proteins, though further investigation is needed to fully elucidate its non-canonical roles .

What is the connection between RPL26L1 and neurological disorders like autism spectrum disorder?

Research has begun to establish connections between RPL26L1 and neurological conditions:

• Biomarker potential: Studies have shown that RPL26L1 is related to autism spectrum disorder and may serve as a potential biomarker for this neurological condition .

• Ribosomal function implications: Given the critical role of proper protein synthesis in neuronal development and function, alterations in RPL26L1 function could impact neurological processes .

• Research approaches: Investigators studying this connection typically employ techniques like RNA-seq to examine expression changes, and antibody-based methods to examine protein levels in patient-derived samples.

While the specific mechanism connecting RPL26L1 to neurological disorders requires further investigation, its role in protein synthesis and potential specialized functions suggest pathways through which dysfunction could contribute to disease states .

What controls should be included when working with RPL26L1 antibodies?

For robust experimental design, include the following controls:

• Positive controls: Cell lines with confirmed RPL26L1 expression such as:

  • HEK-293 cells

  • K-562 cells

  • MCF-7 cells

  • HeLa cells (particularly for IF/ICC)

• Negative controls:

  • Primary antibody omission

  • Isotype control (Rabbit IgG for most available antibodies)

  • Cells with RPL26L1 knockdown/knockout

• Specificity controls:

  • Blocking peptide competition assay using the immunogen peptide

  • Parallel detection with antibodies targeting unique regions of RPL26 versus RPL26L1

• Loading controls: For Western blot, use appropriate loading controls based on your experimental context (e.g., GAPDH, β-actin, or other ribosomal proteins for comparison).

Why might I observe discrepancies between calculated and observed molecular weights for RPL26L1?

Several factors can explain molecular weight discrepancies:

• Post-translational modifications: RPL26L1 has a calculated molecular weight of 17 kDa but typically appears at 22-25 kDa on Western blots , likely due to modifications such as UFMylation .

• UFMylation patterns: Studies have identified mono- and di-UFMylated forms of RPL26L1 that appear as distinct bands (S1 and S2) with increased molecular weights .

• Sample preparation: The buffer conditions and denaturation methods can affect protein migration.

• Gel percentage and running conditions: Higher percentage gels provide better resolution for smaller proteins like RPL26L1.

• Antibody specificity: Some antibodies may detect related proteins or specific modified forms, resulting in unexpected banding patterns.

If observing unexpected molecular weights, researchers should consider these factors and potentially validate their findings using additional approaches such as mass spectrometry or multiple antibodies targeting different epitopes .

How can researchers distinguish between technical artifacts and true biological variations when studying RPL26L1?

To differentiate artifacts from biological variations:

• Use multiple antibodies: Target different epitopes of RPL26L1 to confirm observations. For example, compare results from antibodies targeting N-terminal versus C-terminal regions .

• Employ multiple techniques: Combine Western blot with immunofluorescence or immunohistochemistry to confirm localization and expression patterns .

• Include biological replicates: Test findings across multiple cell lines or tissue samples to ensure reproducibility.

• Genetic validation: Use siRNA knockdown, CRISPR knockout, or overexpression to confirm antibody specificity and biological effects.

• Cross-species validation: If working with conserved pathways, test observations across species using species-specific antibodies .

• Control for post-translational modifications: When studying RPL26L1, consider the impact of modifications like UFMylation that may affect detection and function .

• Validate with non-antibody methods: Use techniques like mass spectrometry or RNA-seq to confirm protein identity and expression levels independently of antibody-based detection .

How can RPL26L1 antibodies be used to study ribosome biogenesis and function?

RPL26L1 antibodies can be powerful tools for ribosome research:

• Ribosome profiling: Use RPL26L1 antibodies to immunoprecipitate ribosomes and study associated mRNAs or proteins in different cellular contexts.

• Subcellular localization: Employ immunofluorescence to track RPL26L1-containing ribosomes, particularly at the endoplasmic reticulum interface where UFMylation appears significant .

• Ribosome assembly: Study the incorporation of RPL26L1 into ribosomal subunits during biogenesis using pulse-chase experiments combined with immunoprecipitation.

• Stress responses: Examine changes in RPL26L1 expression, modification, or localization during cellular stress conditions that affect translation.

• Comparative studies: Investigate the relative incorporation of RPL26 versus RPL26L1 in ribosomes across different tissues or developmental stages to identify specialized functions.

• Quality control mechanisms: Explore the role of RPL26L1 and its UFMylation in translational quality control, particularly for proteins targeted to the endoplasmic reticulum .

Research indicates that RPL26L1's position near the polypeptide exit tunnel makes it particularly relevant for studying co-translational processes and quality control mechanisms .

What emerging research areas might benefit from RPL26L1 antibody applications?

Several cutting-edge research areas could benefit from RPL26L1 studies:

• Specialized ribosomes: Investigate whether RPL26L1 contributes to ribosome heterogeneity and specialized translation in different cellular contexts.

• UFMylation pathway: Further explore the mechanisms and consequences of RPL26L1 UFMylation in cellular quality control processes .

• Neurological disorders: Given the connection to autism spectrum disorder, study RPL26L1 expression and modification in neuronal development and function .

• Stress responses: Examine how cellular stresses affect RPL26L1 incorporation into ribosomes and its post-translational modifications.

• Therapeutic targeting: Investigate whether modulating RPL26L1 function or its modifications might offer therapeutic approaches for disorders involving protein synthesis or quality control defects.

• Single-cell applications: Adapt RPL26L1 antibodies for single-cell analyses to study translation heterogeneity within tissues.