RPL21 Antibody

What applications have been validated for RPL21 antibodies?

RPL21 antibodies have been extensively validated across multiple experimental platforms. Most commercially available antibodies demonstrate reliable performance in Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), and ELISA applications .

Research validation data indicates successful application with the following dilution ranges:

These dilutions should be optimized for each specific experimental system to obtain optimal results, as reactivity can be sample-dependent .

What are the validated species reactivity profiles for RPL21 antibodies?

Most commercial RPL21 antibodies demonstrate cross-reactivity with human, mouse, and rat samples . This conservation reflects the evolutionary importance of ribosomal proteins. Specifically, polyclonal antibodies generated against human RPL21 sequences have shown consistent reactivity across these mammalian species in multiple experimental systems .

When designing experiments using non-validated species, preliminary validation experiments should be conducted to confirm antibody specificity.

What are the key storage and handling protocols for maintaining RPL21 antibody efficacy?

To maintain optimal RPL21 antibody performance, follow these evidence-based storage recommendations:

• Long-term storage: -20°C for up to one year in aliquots to prevent freeze-thaw cycles

• Short-term storage: 4°C for up to one month for frequent use

• Avoid repeated freeze-thaw cycles which can degrade antibody quality

• Most formulations contain 0.02% sodium azide and 50% glycerol at pH 7.2-7.3

• Some preparations include 0.1% BSA in smaller volume preparations

For maximum shelf life, consider aliquoting antibodies into single-use volumes before freezing to minimize freeze-thaw degradation.

What is the expected molecular weight of RPL21 protein in Western blot applications?

RPL21 presents some interesting discrepancies between calculated and observed molecular weights:

This discrepancy between calculated and observed molecular weight likely reflects post-translational modifications or protein-protein interactions that affect migration patterns in SDS-PAGE . When validating a new RPL21 antibody, researchers should expect bands in the 18-25 kDa range, with potential variation based on cell type and experimental conditions.

How has RPL21 been implicated in cancer pathophysiology?

RPL21 demonstrates significant involvement in multiple cancer types, particularly pancreatic and colorectal cancers. Recent research findings include:

• Pancreatic Cancer: RPL21 plays a critical role in pancreatic cancer cell survival and proliferation. siRNA-mediated knockdown of RPL21 significantly suppresses cell proliferation in PANC-1 and BxPC-3 pancreatic cancer cell lines both in vitro and in vivo .

• Colorectal Cancer (CRC): High RPL21 expression correlates with tumor invasiveness and poor patient prognosis. Research demonstrates that RPL21 promotes migration and invasion of CRC cells in vitro and tumor metastasis in vivo .

The significance of RPL21 in these cancer types suggests its potential as both a biomarker and therapeutic target. Immunohistochemical analysis using RPL21 antibodies has been instrumental in establishing these correlations in patient tissue samples .

What molecular mechanisms underlie RPL21's role in cancer cell proliferation?

RPL21 regulates cancer cell proliferation through several interconnected mechanisms:

• Cell Cycle Regulation: RPL21 knockdown induces G1 phase arrest by downregulating key cell cycle regulators including:

  • Cyclin D1 (CCND1)

  • Cyclin E1 (CCNE1)

  • E2F1 transcription factor

• DNA Replication Control: RPL21 positively regulates the mini-chromosome maintenance (MCM) protein family (MCM2-7), which is essential for DNA replication initiation .

• Transcriptional Regulation: Luciferase reporter assays demonstrate that RPL21 controls DNA replication and G1-S phase progression possibly through regulation of E2F1 transcription factor activity in pancreatic cancer cells .

These findings have been validated through multiple experimental approaches, including transcriptome sequencing analysis, qPCR, and Western blotting, using RPL21-specific antibodies to monitor expression levels in various cellular contexts .

How does RPL21 contribute to cancer cell invasion and metastasis?

Recent studies have revealed RPL21's novel role in promoting cancer cell invasion and metastasis, particularly in colorectal cancer:

• LAMP3 Interaction: RPL21 directly binds to LAMP3 (Lysosomal-Associated Membrane Protein 3) through specific protein domains:

  • RPL21 aa 1-40 and aa 111-160 segments bind to the aa 341-416 domain of LAMP3

  • This interaction enhances the stability of RPL21 protein by suppressing ubiquitin-proteasome degradation

• Focal Adhesion Regulation: RPL21 and LAMP3 together promote the formation of immature focal adhesions by activating the FAK/paxillin/ERK signaling pathway, which is critical for cell migration and invasion .

• TFEB Activation: RPL21 activates the transcription factor TFEB, leading to upregulation of LAMP3 expression, creating a positive feedback loop that further enhances invasive potential .

These mechanisms highlight potential therapeutic approaches targeting the RPL21-LAMP3 interaction in metastatic colorectal cancer.

What controls should be included when using RPL21 antibodies in cancer research?

When designing experiments with RPL21 antibodies, include these essential controls:

• Positive Controls:

  • HepG2 cells and HeLa cells for Western blot

  • Human liver tissue for IHC

  • U2OS cells for immunofluorescence

• Negative Controls:

  • Primary antibody omission

  • Non-specific IgG from the same species

  • RPL21 knockdown cells (siRNA-treated) to demonstrate specificity

• Loading Controls:

  • GAPDH or β-Actin for Western blot normalization

• Cell Type Comparisons:

  • Include normal cell lines (e.g., HPDE6-C7 for pancreatic studies) to compare with cancer cells

  • This comparison is particularly relevant as RPL21 siRNA showed apoptosis-inducing effects in cancer cells (BxPC-3, PANC-1) but not in normal cells (HPDE6-C7)

These controls ensure experimental validity and help distinguish between specific and non-specific antibody interactions.

How can RPL21 knockdown experiments be optimized to study its cellular functions?

Based on published research protocols, optimal RPL21 knockdown experimental design includes:

• siRNA Design and Validation:

  • Use a mixture of validated siRNAs targeting different regions of RPL21 mRNA

  • Confirm knockdown efficiency using qPCR and Western blot

  • Research has successfully employed "siL21-Mix" (combining siL21-1 and siL21-2) for effective knockdown

• Functional Assays:

  • Cell proliferation: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays

  • Cell cycle analysis: Flow cytometry with propidium iodide staining

  • Apoptosis assessment: Caspase-8 activity assays and mitochondrial membrane potential measurements

• Gene Expression Analysis:

  • Perform qPCR to validate downstream targets affected by RPL21 knockdown

  • Key genes to monitor include:

    • Upregulated: AHR, THBS1, DDIT3, MKNK2

    • Downregulated: E2F1, PCNA, CCND1, CCNE1, MCM2, MCM4, MCM5, MCM7, KIAA0101

• In Vivo Validation:

  • BALB/c nude mice tumor models have been successfully used to translate in vitro findings to in vivo contexts

This comprehensive approach ensures robust evaluation of RPL21's biological functions.

What technical considerations should be addressed when performing immunohistochemistry with RPL21 antibodies?

Optimizing IHC with RPL21 antibodies requires attention to these technical parameters:

• Antigen Retrieval:

  • Recommended protocol: TE buffer pH 9.0

  • Alternative protocol: Citrate buffer pH 6.0

  • Antigen retrieval is critical as RPL21 may have epitopes masked by formalin fixation

• Antibody Dilution:

  • Initial range: 1:20-1:200

  • Precise dilution should be determined through titration experiments

• Detection Systems:

  • HRP-conjugated secondary antibodies provide good sensitivity

  • Consider amplification systems for low-abundance detection

• Counterstaining and Visualization:

  • Nuclear counterstain helps contextualize RPL21 localization

  • RPL21 typically shows both cytoplasmic and nuclear localization patterns

• Quantification Methods:

  • Develop standardized scoring systems based on staining intensity and distribution

  • Consider digital image analysis for more objective quantification

These methodological considerations ensure consistent and reproducible IHC results with RPL21 antibodies.

How can RPL21 antibodies be utilized to investigate ribosomal protein moonlighting functions?

Ribosomal proteins, including RPL21, perform functions beyond their classical role in protein synthesis. RPL21 antibodies can help investigate these moonlighting functions through:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation experiments to identify novel binding partners

  • Proximity ligation assays to visualize and confirm interactions in situ

  • Recent research has already identified LAMP3 as a non-canonical binding partner

• Subcellular Localization Analysis:

  • Immunofluorescence microscopy to track RPL21 localization under different cellular conditions

  • Fractionation studies combined with Western blotting to quantify RPL21 distribution

• Chromatin Association Analysis:

  • Chromatin immunoprecipitation (ChIP) to investigate potential direct or indirect DNA binding

  • This approach may elucidate RPL21's role in transcriptional regulation via E2F1

These approaches can reveal novel functions of RPL21 beyond ribosome biogenesis and protein synthesis.

What are the potential therapeutic implications of targeting RPL21 in cancer?

Research findings suggest several therapeutic strategies targeting RPL21:

• RNA Interference Approaches:

  • siRNA against RPL21 has demonstrated anti-cancer activity in pancreatic cancer models

  • Effects include proliferation inhibition, DNA replication suppression, G1 cell cycle arrest, and apoptosis induction

• Protein-Protein Interaction Inhibitors:

  • The interaction between RPL21 and LAMP3 represents a potential therapeutic target

  • Small molecule inhibitors disrupting this interaction could reduce metastatic potential in colorectal cancer

• Cancer-Specific Effects:

  • RPL21 knockdown induces apoptosis in cancer cells but not in normal cells, suggesting a therapeutic window

  • This differential effect could be exploited to develop cancer-specific treatments

• Biomarker Potential:

  • RPL21 expression correlates with poor prognosis in colorectal cancer

  • RPL21 antibodies could be used in diagnostic and prognostic applications

These approaches highlight RPL21's potential as both a therapeutic target and biomarker in cancer management.

How can non-specific binding be minimized when using RPL21 antibodies?

To reduce non-specific binding in experimental applications:

• Blocking Optimization:

  • For Western blotting: 3-5% non-fat dry milk in TBST has shown good results

  • For IHC/IF: BSA or serum from the same species as the secondary antibody

• Antibody Selection:

  • Use affinity-purified antibodies when possible

  • Proteintech's 15226-1-AP and Biorbyt's orb1260028 antibodies have been validated for specificity

• Dilution Optimization:

  • Titrate antibodies to find the optimal concentration that maximizes specific signal while minimizing background

  • Different applications require different optimal dilutions

• Washing Procedures:

  • Implement more stringent washing steps (increased duration or number of washes)

  • Consider adding low concentrations of detergent (0.05-0.1% Tween-20) to wash buffers

• Pre-adsorption Controls:

  • Pre-incubate the antibody with recombinant RPL21 protein to confirm binding specificity

These approaches can significantly improve signal-to-noise ratio in RPL21 antibody applications.

What are common discrepancies in experimental results with RPL21 antibodies and how can they be addressed?

Researchers may encounter these common discrepancies when working with RPL21 antibodies:

• Molecular Weight Variations:

  • Expected: 18-19 kDa (calculated)

  • Observed: 20-25 kDa range

  • Solution: Use positive control lysates (HepG2, HeLa) to establish expected band pattern

• Differential Cellular Localization:

  • Nuclear vs. cytoplasmic distribution can vary by cell type and physiological state

  • Solution: Include subcellular fractionation controls and co-staining with organelle markers

• Variable Expression Levels:

  • RPL21 expression can vary significantly between cell types and cancer models

  • Solution: Normalize to housekeeping genes (GAPDH, β-Actin) and include appropriate tissue controls

• Batch-to-Batch Variations:

  • Polyclonal antibodies may show lot-to-lot variation

  • Solution: Validate each new lot against previous lots using consistent positive controls

• Cross-Reactivity with Related Proteins:

  • Other ribosomal proteins may share sequence homology

  • Solution: Confirm specificity using RPL21 knockdown controls

Addressing these issues systematically ensures more reliable and reproducible results when working with RPL21 antibodies.