RPS6KL1 Antibody

What is RPS6KL1 and why is it relevant for research?

RPS6KL1 (Ribosomal Protein S6 Kinase-Like 1) is a 549 amino acid protein belonging to the Ser/Thr protein kinase family, S6 kinase subfamily, and protein kinase superfamily. It contains one MIT domain and a protein kinase domain and exists in three alternatively spliced isoforms. The gene encoding RPS6KL1 maps to human chromosome 14, which houses over 700 genes and comprises nearly 3.5% of the human genome . While its exact function remains under investigation, its relationship to other S6 kinases suggests potential roles in cellular signaling pathways related to protein synthesis and cell growth regulation.

What types of RPS6KL1 antibodies are available and how should I choose between them?

Several types of RPS6KL1 antibodies are available, varying in host species, clonality, and target epitopes:

Selection should be based on your specific application, target species, and epitope of interest. For example, antibodies targeting amino acids 1-541 recognize the full-length protein , while those targeting specific regions may be useful for detecting particular isoforms or post-translational modifications. Consider validated applications (WB, IHC, ELISA, IF) when selecting an antibody for your experiments .

What species reactivity should I consider when selecting an RPS6KL1 antibody?

Available RPS6KL1 antibodies show reactivity with various species:

Always verify the species reactivity for your target model system. While many antibodies are validated for human samples, fewer are confirmed for mouse and rat models. Some antibodies demonstrate cross-reactivity with multiple species due to sequence conservation .

What are the optimal working dilutions for different applications of RPS6KL1 antibodies?

Recommended dilutions vary by application and specific antibody:

Always perform titration experiments to determine optimal dilutions for your specific experimental conditions. Antibody performance can vary between lots and across different cell or tissue types .

How should I prepare samples for optimal detection of RPS6KL1 using antibodies?

For reliable RPS6KL1 detection, consider these sample preparation protocols:

• Western Blotting:

  • Use RIPA or NP-40 buffer with protease and phosphatase inhibitors

  • Recommended protein loading: 20-25 μg per lane

  • Reducing conditions are typically required for optimal detection

  • Separate proteins on 8-12% SDS-PAGE gels (RPS6KL1 runs at approximately 60-70 kDa)

• Immunohistochemistry:

  • Fixation: 10% neutral buffered formalin (24-48 hours)

  • Antigen retrieval: Citrate buffer (pH 6.0) heat-induced epitope retrieval

  • Blocking: 3-5% normal serum (matching secondary antibody host)

  • Primary antibody incubation: Overnight at 4°C

• Immunofluorescence:

  • Fixation: 4% paraformaldehyde (10-15 minutes)

  • Permeabilization: 0.1-0.5% Triton X-100 in PBS (10 minutes)

  • Blocking: 1-5% BSA in PBS (30-60 minutes)

What controls should I include when using RPS6KL1 antibodies?

Include these controls for reliable RPS6KL1 antibody experiments:

• Positive Controls:

  • Cell lines with confirmed RPS6KL1 expression (e.g., MCF-7 breast cancer cells, A549 cells)

  • Human tissue samples known to express RPS6KL1 (e.g., testis, colon cancer tissue)

• Negative Controls:

  • Primary antibody omission control

  • Isotype control (rabbit or mouse IgG depending on host species)

  • siRNA knockdown or CRISPR knockout of RPS6KL1 (for specificity validation)

• Loading Controls (for Western blotting):

  • GAPDH, β-actin, or α-tubulin to normalize protein loading

• Peptide Competition Assay:

  • Pre-incubation of antibody with immunizing peptide to confirm specificity

How can I verify the specificity of my RPS6KL1 antibody results?

Validate antibody specificity through multiple approaches:

• Molecular Weight Verification:

  • RPS6KL1 should appear at approximately 60-70 kDa on Western blots

  • Be aware of potential post-translational modifications that may alter migration patterns

• Multiple Antibody Approach:

  • Use antibodies targeting different epitopes of RPS6KL1

  • Concordant results across different antibodies increase confidence in specificity

• Genetic Validation:

  • siRNA or shRNA knockdown of RPS6KL1

  • CRISPR/Cas9 knockout of RPS6KL1

  • Overexpression of RPS6KL1 with epitope tags

• Peptide Competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Specific staining should be abolished or significantly reduced

What are potential cross-reactivity concerns with RPS6KL1 antibodies?

RPS6KL1 antibodies may cross-react with:

• Related Kinase Family Members:

  • Other S6 kinase family proteins (RPS6KA1, RPS6KB1, etc.) due to sequence homology

  • P70 S6 kinase shares structural similarities and may be detected by some antibodies

• Isoforms and Splice Variants:

  • RPS6KL1 exists in multiple isoforms that may not all be recognized by a single antibody

  • Antibodies targeting specific regions may miss certain splice variants

• Minimizing Cross-Reactivity:

  • Select antibodies validated for your specific application and species

  • Consider using antibodies raised against unique regions of RPS6KL1 with minimal homology to related proteins

How should I interpret RPS6KL1 expression patterns in different tissues and experimental conditions?

When interpreting RPS6KL1 expression:

• Tissue-Specific Expression:

  • RPS6KL1 shows different expression patterns across tissues

  • Testis and colon cancer tissues have been reported to express detectable RPS6KL1 levels

• Subcellular Localization:

  • Immunofluorescence studies suggest RPS6KL1 may have both cytoplasmic and nuclear distribution patterns

  • Subcellular localization may change under different cellular conditions

• Expression Changes:

  • Compare with appropriate controls (normal vs. pathological tissue, treated vs. untreated cells)

  • Quantify relative expression changes using densitometry for Western blots or fluorescence intensity measurements for IF/IHC

• Variability Sources:

  • Technical variability (antibody lot, protocol differences)

  • Biological variability (donor differences, cell culture conditions)

  • Consider statistical analysis of multiple biological replicates

How can I use RPS6KL1 antibodies in studying signaling pathways related to cancer?

Recent research suggests connections between S6 kinase family members and cancer pathways:

• Therapeutic Resistance Studies:

  • The related kinase RPS6KA1 has been implicated in resistance to venetoclax/azacitidine therapy in acute myeloid leukemia (AML)

  • RPS6KA1 inhibition may sensitize resistant cells to treatment

• Experimental Approaches:

  • Phospho-specific antibodies can track activation status of RPS6KL1 and related kinases

  • Co-immunoprecipitation with RPS6KL1 antibodies can identify interaction partners

  • Combine with kinase inhibitors to study pathway dependence

• Combination with Other Techniques:

  • Use RPS6KL1 antibodies in ChIP-seq to identify potential transcriptional targets

  • Combine with phospho-proteomic approaches to map signaling networks

What methodological considerations are important when using RPS6KL1 antibodies in multiplex assays?

For multiplex applications with RPS6KL1 antibodies:

• Antibody Compatibility:

  • Select antibodies from different host species to avoid cross-reactivity of secondary antibodies

  • For same-species antibodies, use directly conjugated primary antibodies or sequential staining protocols

• Signal Separation:

  • Choose fluorophores with minimal spectral overlap

  • Include appropriate single-stain controls for compensation/unmixing

  • Set proper exposure times to avoid bleed-through

• Antibody Validation for Multiplex:

  • Validate each antibody individually before combining

  • Compare multiplex results with single-plex to ensure signal fidelity

  • Include appropriate blocking steps between sequential stainings

How can I investigate the functional relationships between RPS6KL1 and other S6 kinase family members?

To study functional relationships between RPS6KL1 and related kinases:

• Comparative Expression Analysis:

  • Use antibodies specific for different family members (RPS6KA1, RPS6KB1, RPS6KL1)

  • Compare expression patterns across tissues and conditions

  • Analyze co-expression or mutually exclusive expression patterns

• Pathway Interaction Studies:

  • Use combined immunoprecipitation and Western blot to detect interactions

  • Study phosphorylation cascades using phospho-specific antibodies

  • Investigate substrate overlap through phospho-proteomic approaches

• Functional Redundancy Assessment:

  • Perform selective knockdown of individual family members

  • Analyze compensatory changes in expression or activity

  • Test the effects of selective inhibitors on downstream pathways

What are common issues when using RPS6KL1 antibodies and how can they be resolved?

Common challenges and solutions:

How can I optimize detection of low-abundance RPS6KL1 in challenging samples?

For detecting low-abundance RPS6KL1:

• Sample Enrichment:

  • Consider subcellular fractionation to concentrate protein from relevant compartments

  • Immunoprecipitation before Western blotting to enrich RPS6KL1

  • Use tissue samples with known higher expression (e.g., testis)

• Signal Amplification:

  • For IHC/IF: Use tyramide signal amplification (TSA) systems

  • For Western blot: Consider high-sensitivity chemiluminescent substrates

  • Longer exposure times balanced against background increase

• Protocol Optimization:

  • Reduce washing stringency slightly (lower salt, less detergent)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal enhancers compatible with your detection system

How should I approach contradictory results between different RPS6KL1 antibodies?

When faced with discrepant results:

• Epitope Mapping:

  • Determine exact binding regions of each antibody

  • Different epitopes might be masked in certain protein conformations or complexes

  • Some epitopes may be removed by proteolytic processing

• Post-Translational Modification Analysis:

  • Investigate if modifications (phosphorylation, ubiquitination) affect antibody binding

  • Use phosphatase treatment to determine if phosphorylation impacts detection

• Validation Approach:

  • Perform knockdown/knockout experiments with each antibody

  • Use orthogonal methods to verify expression (qPCR, mass spectrometry)

  • Consider antibody-independent methods (e.g., CRISPR tagging of endogenous protein)

How can RPS6KL1 antibodies be utilized in single-cell analysis techniques?

Applications in single-cell research:

• Single-Cell Western Blotting:

  • Microfluidic platforms allow protein analysis at single-cell level

  • Requires optimization of RPS6KL1 antibodies for lower protein amounts

  • Can reveal cell-to-cell variability in expression levels

• Mass Cytometry (CyTOF):

  • Metal-conjugated RPS6KL1 antibodies can be used in CyTOF panels

  • Enables simultaneous detection of multiple proteins in single cells

  • Can correlate RPS6KL1 expression with cell type and functional markers

• Optimization Considerations:

  • Higher antibody specificity requirements for single-cell applications

  • Validation using knockout controls is essential

  • Signal amplification may be necessary for detection of low-abundance targets

What are promising research directions involving RPS6KL1 in disease mechanisms?

Emerging research areas:

• Cancer Research:

  • Related kinases (RPS6KA1) have been implicated in therapeutic resistance in AML

  • The role of RPS6KL1 in cancer cell signaling remains to be fully elucidated

  • Potential therapeutic target or biomarker for certain cancer types

• Neurodevelopmental Disorders:

  • The gene encoding RPS6KL1 maps to human chromosome 14, which has been associated with certain neurological conditions

  • Further research needed to establish potential functional roles

• Cell Signaling Regulation:

  • As a member of the S6 kinase family, RPS6KL1 may participate in translation regulation

  • Understanding its interactome and substrates could reveal novel regulatory mechanisms

How might phospho-specific RPS6KL1 antibodies advance our understanding of its activation and regulation?

The development and application of phospho-specific antibodies could:

• Identify Activation Mechanisms:

  • Track phosphorylation events that regulate RPS6KL1 activity

  • Map kinase cascades that lead to RPS6KL1 activation

  • Study temporal dynamics of activation in response to stimuli

• Therapeutic Monitoring:

  • Assess efficacy of kinase inhibitors on pathway activity

  • Monitor treatment responses at the cellular level

  • Identify resistance mechanisms through altered phosphorylation patterns

• Cross-talk with Other Pathways:

  • Investigate how RPS6KL1 phosphorylation status changes with other signaling events

  • Study potential convergence of multiple pathways on RPS6KL1 regulation

  • Understand compensatory mechanisms when related kinases are inhibited