RPL26 Antibody, Biotin conjugated

What is RPL26 and why is it important in research?

RPL26 (ribosomal protein L26) is a component of the 60S large ribosomal subunit with a calculated molecular weight of 17 kDa, though it is often observed at 17-22 kDa in experimental conditions. Beyond its structural role in ribosomes, RPL26 has gained significant research interest due to its involvement in p53 mRNA translation regulation following cellular stress. RPL26 interacts with nucleolin and binds to a double-stranded RNA structure in p53 mRNA to enhance p53 translation, particularly after DNA damage . This regulatory mechanism represents an important layer of post-transcriptional control in the stress response pathway, making RPL26 a valuable target for cancer research and cellular stress studies .

What is the difference between RPL26 and RPL26L1?

RPL26 is the canonical ribosomal protein L26, while RPL26L1 (also known as RPL26P1) is the L26-like 1 variant or paralog . While both are components of the 60S ribosomal subunit, they have distinct gene locations and potentially different functional specializations. RPL26L1 is involved in several cellular processes including cytoplasmic translation, gene expression, nuclear-transcribed mRNA catabolic processes, nonsense-mediated decay, and ribosomal large subunit biogenesis . RPL26 has been extensively studied for its role in p53 regulation following cellular stress, while the specific functions of RPL26L1 that differentiate it from RPL26 are still being elucidated. When selecting antibodies, researchers should be careful to choose the specific target (RPL26 or RPL26L1) relevant to their research question .

What are the standard formats of RPL26 antibodies available?

RPL26 antibodies are available in several formats, with unconjugated being the most common for applications like Western blotting, immunohistochemistry, and immunoprecipitation . Biotin-conjugated antibodies, like the RPL26L1 polyclonal antibody (A68125-050), offer advantages for certain detection systems, particularly ELISA . The conjugation status affects the antibody's application range, with unconjugated versions generally being more versatile across multiple techniques. Most commercially available RPL26 antibodies are polyclonal and raised in rabbits (rabbit IgG), though monoclonal options may also be available . The storage format typically includes PBS with glycerol (often 50%) and preservatives like sodium azide (0.02%) or Proclin 300 (0.03%) to maintain stability during long-term storage at -20°C or -80°C .

What are the validated applications for RPL26 antibodies?

The RPL26 antibody (17619-1-AP) has been validated for multiple applications including Western Blot (WB), Immunofluorescence (IF)/Immunocytochemistry (ICC), Immunohistochemistry (IHC), Co-Immunoprecipitation (CoIP), and ELISA . For Western blotting, the recommended dilution is 1:500-1:1000, while for IF/ICC applications, a dilution range of 1:50-1:500 is suggested . The antibody has demonstrated positive WB results in several cell lines including K-562, HeLa, HepG2, and Jurkat cells, while positive IF/ICC results have been confirmed in HeLa cells . Published literature has documented successful use of RPL26 antibodies in knockout/knockdown studies, as well as in multiple applications including CoIP, which is particularly relevant for studying the interaction between RPL26 and nucleolin . The biotin-conjugated RPL26L1 antibody (A68125-050) is specifically validated for ELISA applications .

How should biotin-conjugated RPL26 antibodies be used in ELISA protocols?

For ELISA applications using biotin-conjugated RPL26L1 antibody, researchers should follow standard avidin-biotin detection systems. The biotin conjugation enables detection through streptavidin or avidin coupled to enzymes like horseradish peroxidase (HRP) or alkaline phosphatase. A typical protocol would involve coating plates with capture antibody or antigen, blocking, adding samples, followed by the biotin-conjugated RPL26L1 antibody as a detection antibody, and finally adding streptavidin-HRP for signal development. The protein G-purified nature of the antibody (A68125-050) ensures high specificity and low background . Researchers should determine optimal dilutions empirically, though ELISAs typically use antibody dilutions in the range of 1:1,000 to 1:10,000. The antibody's buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) should be considered when calculating final concentrations and when determining compatibility with the ELISA system being used .

What methods are recommended for co-immunoprecipitation studies with RPL26?

Co-immunoprecipitation (CoIP) studies with RPL26 antibodies require careful consideration of buffer conditions and experimental design. Based on published protocols, cells should be lysed in appropriate buffers such as radioimmune precipitation assay (RIPA) buffer or TGN buffer . For studying the interaction between RPL26 and nucleolin, precleared lysates (1 mg of whole cell extract) should be incubated overnight with the appropriate antibodies, such as anti-RPL26 or anti-nucleolin . Following extensive washing with lysis buffer, immunoprecipitated proteins can be analyzed by Western blotting. Primary antibodies used for detection may include anti-p53 (DO-1), anti-nucleolin (MS-3), anti-GFP (for tagged constructs), anti-FLAG (M2), anti-RPL26, or anti-actin . Secondary antibodies would typically be HRP-conjugated anti-rabbit or anti-mouse, with visualization using an ECL system . This approach has successfully demonstrated the interaction between RPL26 and nucleolin and their roles in p53 mRNA regulation.

How does RPL26 regulate p53 translation and what are the implications for cancer research?

RPL26 plays a critical role in regulating p53 translation, particularly following cellular stress such as DNA damage. The mechanism involves RPL26 binding to a double-stranded RNA structure formed by complementary sequences in the 5′ and 3′ untranslated regions (UTRs) of p53 mRNA . This binding enhances p53 translation, contributing to p53 protein induction after stress. Interestingly, nucleolin (NCL) acts antagonistically to RPL26 by suppressing p53 translation under normal conditions . Both proteins interact with each other and compete for binding to the same region in p53 mRNA. After DNA damage, this balance shifts, with RPL26 disrupting nucleolin dimerization and promoting p53 translation . For cancer research, this mechanism represents a potential therapeutic target, as many cancers show dysregulation of p53 pathways. Understanding the RPL26-nucleolin axis could lead to strategies that manipulate p53 levels post-transcriptionally, potentially restoring normal p53 function in cancer cells with wild-type p53 .

What experimental approaches can distinguish between the functions of RPL26 and RPL26L1?

Distinguishing the specific functions of RPL26 and its paralog RPL26L1 requires comprehensive experimental approaches. Researchers can employ knockdown or knockout studies using siRNA, shRNA, or CRISPR-Cas9 systems targeting each specific gene to observe differential phenotypic effects. Using antibodies specific to each protein (such as the 17619-1-AP for RPL26 and A68125-050 for RPL26L1) in Western blotting and immunoprecipitation can help identify unique binding partners and post-translational modifications . RNA-sequencing following individual knockdowns can reveal distinct transcriptional networks affected by each protein. Importantly, functional rescue experiments where one protein is depleted and then either RPL26 or RPL26L1 is reintroduced can definitively demonstrate unique versus redundant functions. For studying p53 regulation specifically, reporter assays using p53 5′ and 3′ UTR constructs can determine whether RPL26L1 shares RPL26's ability to enhance p53 translation following cellular stress . These complementary approaches can together establish the unique contribution of each protein to cellular processes.

What is the significance of the RNA-binding domain of nucleolin in its interaction with RPL26?

The RNA-binding domain (RBD) of nucleolin plays a crucial role in the regulatory mechanism involving RPL26 and p53 mRNA. Research has shown that this domain is indispensable for nucleolin binding to p53 mRNA, nucleolin self-interaction (dimerization), nucleolin-RPL26 interaction, and nucleolin-dependent translational repression . The RBD allows nucleolin to simultaneously and independently bind to both RNA sequences of the base-pairing interaction region in p53 mRNA, the same region that is critical for RPL26's translational enhancement effects . Nucleolin appears to stabilize the double-stranded RNA structure in this region under normal conditions, thereby repressing p53 translation. Following cellular stress, RPL26 can disrupt nucleolin dimerization, suggesting a model where the competition between nucleolin-nucleolin homodimers and RPL26-nucleolin interactions serves as a molecular switch between translational repression and activation . Mutations in the NCL-interacting region of RPL26 reduce this interaction and attenuate both RPL26 binding to p53 mRNA and p53 induction, highlighting the importance of this domain-specific interaction in the stress response pathway .

What are common issues when using biotin-conjugated antibodies in multicolor flow cytometry?

When using biotin-conjugated antibodies like RPL26L1 in multicolor flow cytometry, several technical challenges may arise. One common issue is the potential for cross-reactivity with endogenous biotin in samples, which can produce high background signals. This can be mitigated by using biotin-blocking kits before adding the biotin-conjugated antibody. Another challenge is the large size of streptavidin-fluorophore complexes, which may cause steric hindrance and affect binding to the target epitope. Researchers should also be aware that the strong biotin-streptavidin interaction makes these conjugates difficult to strip for sequential staining protocols. Signal amplification through the avidin-biotin system can be both an advantage and a disadvantage, potentially producing oversaturated signals that require careful titration of the antibody. For RPL26/RPL26L1 specifically, researchers should consider that these are predominantly intracellular proteins, necessitating proper cell permeabilization protocols to allow antibody access . Optimization steps should include careful antibody titration, appropriate controls (including FMO - Fluorescence Minus One), and validation using cells with known expression levels of the target protein.

How can specificity of RPL26 antibodies be validated in experimental systems?

Validating the specificity of RPL26 antibodies is crucial for accurate experimental results. A comprehensive validation approach should include several complementary methods. Western blotting using cell lysates from multiple cell lines (such as K-562, HeLa, HepG2, and Jurkat cells for RPL26) can confirm the presence of a band at the expected molecular weight (17-22 kDa for RPL26) . Knockout or knockdown validation is particularly important, where the antibody signal should be absent or significantly reduced in cells where RPL26 expression has been eliminated or suppressed using CRISPR-Cas9, siRNA, or shRNA techniques . Immunoprecipitation followed by mass spectrometry can confirm that the antibody is indeed pulling down RPL26 rather than cross-reacting with similar proteins. For RPL26, given its similarity to RPL26L1, researchers should specifically check for cross-reactivity between these paralogs. Pre-absorption tests, where the antibody is pre-incubated with purified recombinant RPL26 protein before application, should eliminate specific staining if the antibody is truly specific. Finally, multiple antibodies targeting different epitopes of RPL26 should show similar staining patterns, providing further validation of specificity .

What are the critical parameters for optimizing immunofluorescence with RPL26 antibodies?

Optimizing immunofluorescence staining with RPL26 antibodies requires attention to several critical parameters. First, fixation method significantly impacts epitope accessibility and preservation - for RPL26, which has been successfully detected in HeLa cells, paraformaldehyde fixation (typically 4%) for 10-15 minutes at room temperature is commonly effective . The permeabilization step is crucial since RPL26 is primarily localized in the cytosol and cytosolic large ribosomal subunit; Triton X-100 (0.1-0.5%) or methanol permeabilization may be used, with optimization needed for each cell type . Antibody dilution requires careful titration, with a recommended starting range of 1:50-1:500 for the RPL26 antibody (17619-1-AP) . Incubation conditions should be optimized, typically starting with overnight incubation at 4°C for primary antibodies. For biotin-conjugated antibodies, a dedicated blocking step for endogenous biotin is advisable before antibody application. Appropriate controls must include a negative control omitting primary antibody and a peptide competition assay. Signal amplification methods should be considered if the target shows low expression, with biotin-conjugated secondary antibodies followed by fluorophore-conjugated streptavidin potentially offering enhanced sensitivity. Finally, counterstaining of subcellular compartments (e.g., DAPI for nuclei, phalloidin for actin) can provide context for RPL26 localization .

What are emerging research directions involving RPL26 beyond p53 regulation?

While RPL26's role in p53 translation regulation is well-established, emerging research is expanding our understanding of its functions beyond this pathway. RPL26 is increasingly recognized as a multifunctional protein potentially involved in diverse cellular processes including ribosome biogenesis, nucleolar stress response, and regulation of other mRNAs with structured UTRs similar to p53 . Its interaction with nucleolin suggests potential roles in broader RNA metabolism networks, possibly extending to other tumor suppressors or oncogenes . The evolutionary conservation of RPL26 across species indicates fundamental biological importance beyond its specialized role in p53 regulation. Recent publications have begun exploring connections between RPL26 and other cellular stress responses beyond DNA damage, including endoplasmic reticulum stress, oxidative stress, and nutrient deprivation . Additionally, the relationship between RPL26 and its paralog RPL26L1 remains an area ripe for investigation, with potential implications for understanding ribosomal protein gene duplication and functional specialization . Future research might also explore post-translational modifications of RPL26 and how these affect its extra-ribosomal functions, interaction partners, and subcellular localization under different physiological and pathological conditions.