RPS12 Antibody

What is RPS12 and what cellular functions does it perform?

RPS12 (ribosomal protein S12) is a component of the 40S ribosomal subunit involved in the small subunit (SSU) processome. It functions as part of the first precursor of the small eukaryotic ribosomal subunit. During SSU processome assembly in the nucleolus, RPS12 works with other ribosome biogenesis factors and RNA chaperones to facilitate RNA folding, modifications, rearrangements, and cleavage . Together with ribosomal proteins S4 and S5, RPS12 plays a critical role in translational accuracy . The protein has a molecular weight of approximately 14-15 kDa , with slight variations depending on the species. In addition to its role in translation, research has demonstrated that RPS12 is essential for embryonic development and hematopoietic stem cell maintenance .

Mouse models with heterozygous deletion of RPS12 (Rps12 KO/+) exhibit phenotypes that inform our understanding of ribosome-related diseases like Diamond-Blackfan Anemia (DBA):

• Rps12 KO/+ mice display reduced body size, morphological defects, and in some cases hydrocephalus

• These mice present with pancytopenia (reduction in all blood cell types)

• A striking reduction in hematopoietic stem cell (HSC) populations is observed:

  • Long-term HSCs (LT-HSCs: Flk2-CD48-CD150+Lineage-Sca1+c-kit+)

  • Short-term HSCs (ST-HSCs: Flk2-CD48-CD150-LSK)

• Decreased bone marrow cellularity and reduced spleen size

• Increased apoptosis in bone marrow cells, particularly in the Lineage-Sca1+c-Kit+ (LSK) population containing HSCs and multipotent progenitors

These findings demonstrate that RPS12 has essential roles beyond protein synthesis, specifically in maintaining hematopoietic stem cell viability and function. Interestingly, while homozygous deletion of Rps12 is embryonically lethal, heterozygous models show specific defects in hematopoiesis, making them valuable for studying mechanisms of ribosomopathies .

What are the optimal protocols for Western blot detection of RPS12?

For optimal Western blot detection of RPS12, researchers should follow these methodological guidelines:

• Sample preparation:

  • Extract proteins using standard lysis buffers (e.g., Tris-HCl pH 6.8 50mM, 10mM EDTA, 2% SDS with protease inhibitors)

  • Load 10-25 μg of total protein per lane

• Electrophoresis and transfer:

  • Use 15% SDS-PAGE gels to effectively resolve the 14-15 kDa RPS12 protein

  • Perform wet transfer to nitrocellulose membranes for best results

• Antibody incubation:

  • Block membranes in TBS-T containing 5% non-fat dry milk for 1 hour at room temperature

  • Dilute primary antibody 1:1000-1:4000 in blocking buffer

  • Incubate for 1-2 hours at room temperature or overnight at 4°C

• Detection:

  • Use HRP-conjugated secondary antibodies (typically at 1:10,000 dilution)

  • Visualize using standard ECL detection methods

• Positive controls:

  • Validated cell lines include NIH/3T3, HeLa, MCF-7, and HepG2

The observed molecular weight on Western blots is typically 15 kDa, consistent with the calculated molecular weight (14.5-15 kDa) .

How do RPS12 antibodies perform in immunofluorescence applications?

For immunofluorescence detection of RPS12, researchers should consider the following methodological approach:

• Sample preparation:

  • Cells: HepG2, HeLa, A549, and MCF-7 cells have been validated for RPS12 immunofluorescence

  • Fix with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100

• Antibody dilution and incubation:

  • Use 1:200-1:800 dilution of primary antibody

  • For plant/algal samples, RPS12 chloroplastic antibodies may require different dilutions (1:10,000 recommended for certain applications)

• Visualization:

  • Secondary antibodies conjugated with fluorophores

  • DAPI is commonly used for nuclear counterstaining

• Expected localization:

  • Cytoplasmic distribution with enrichment in the nucleolus for mammalian RPS12

  • Chloroplastic distribution for plant RPS12

Confocal microscopy provides higher resolution for visualizing the subcellular localization of RPS12, particularly for distinguishing nucleolar localization from cytoplasmic ribosomal components .

What changes occur in global translation following RPS12 depletion?

Research on RPS12 haploinsufficiency has revealed complex and time-dependent effects on protein translation:

• Acute deletion effects: When RPS12 is acutely deleted in adult hematopoietic cells using an inducible Cre-lox system, global translation initially decreases

• Chronic adaptation effects: In contrast, mice that are heterozygous for RPS12 from fertilization show an unexpected increase in global protein translation, particularly in hematopoietic stem cells and multipotent progenitors

• Cell-type specific effects: The impact on translation varies between different cell populations:

  • HSCs show the most dramatic increase in translation

  • Myeloid progenitors generally do not exhibit significant changes in translation rates

  • Megakaryocyte-erythrocyte progenitors (MEP) show a significant increase in translation

These contradictory findings suggest a complex compensatory mechanism in response to chronic RPS12 deficiency. The increased translation in HSCs may contribute to their depletion through proteotoxic stress and increased apoptosis . These observations underscore the importance of temporal considerations when studying ribosomal protein functions in vivo.

What are the distinct features of chloroplastic RPS12 antibodies compared to cytoplasmic versions?

Chloroplastic RPS12 antibodies differ from those targeting cytoplasmic RPS12 in several important aspects:

When designing experiments involving plant systems, researchers should select chloroplastic-specific RPS12 antibodies to accurately target the plastid ribosomal protein . Immunogen design is typically based on recombinant full-length RPS12 from model organisms like Chlamydomonas reinhardtii .

How do conditional knockout models of RPS12 contribute to our understanding of its function?

Conditional knockout models have provided critical insights into the tissue-specific and temporal functions of RPS12:

• Germline models:

  • Homozygous knockout (Rps12 KO/KO) is embryonically lethal, demonstrating the essential nature of RPS12

  • Heterozygous knockout (Rps12 KO/+) mice are viable but display reduced body size, morphological defects, and hematopoietic abnormalities

• Inducible hematopoietic-specific models:

  • Rps12 flox/+; Tal1-Cre-ERT system allows tamoxifen-induced deletion specifically in hematopoietic cells

  • Acute deletion of RPS12 in adult hematopoietic cells leads to:

    • No significant changes at 7 weeks post-excision

    • Progressive development of hematopoietic defects by 12-14 weeks

    • Different translation phenotypes compared to germline heterozygotes

• Generation technique:

  • CRISPR/Cas9-based gene editing with guide RNAs targeting introns 1 and 3

  • Homology-directed repair incorporating loxP sites flanking exons 2 and 3

These models enable the distinction between developmental versus adult-specific functions of RPS12 and reveal tissue-specific requirements for this ribosomal protein. The different outcomes between germline and inducible models highlight the importance of developmental timing in ribosomal protein function .

What experimental considerations are important when using RPS12 antibodies across different species?

When conducting cross-species studies with RPS12 antibodies, researchers should consider:

• Cross-reactivity validation:

  • Confirmed reactivity: Human, mouse, rat for cytoplasmic RPS12 antibodies

  • Chloroplastic RPS12 antibodies: Arabidopsis thaliana, Chlamydomonas reinhardtii, Zea mays

  • Predicted but unconfirmed reactivity should be experimentally validated

• Species-specific protocols:

  • Plant samples may require different extraction buffers than mammalian samples

  • Dilution requirements may vary between species even with confirmed reactivity

• Protein homology considerations:

  • High sequence conservation among mammals (100% sequence identity predicted between human, mouse, rat, bovine, and pig)

  • Greater divergence between mammalian and plant/bacterial RPS12

• Application limitations:

  • Species-application combinations validated for Western blot may not work for immunohistochemistry

  • Non-validated combinations should be tested with appropriate positive and negative controls

• Isoform specificity:

  • Confirm which isoform of RPS12 the antibody recognizes if multiple exist in the target species

  • Consider whether the antibody recognizes post-translational modifications

Researchers should review validation data specific to their species of interest and application before proceeding .

How can researchers analyze RPS12 involvement in hematopoietic stem cell function?

Based on recent findings regarding RPS12's role in hematopoietic stem cell (HSC) function, researchers can employ the following experimental approaches:

• Flow cytometry analysis of stem and progenitor populations:

  • Long-term HSCs (LT-HSCs): Flk2-CD48-CD150+Lineage-Sca1+c-kit+

  • Short-term HSCs (ST-HSCs): Flk2-CD48-CD150-LSK

  • Multipotent progenitors (MPPs) and other progenitor populations

• Global translation analysis:

  • Ex vivo assay using o-propargyl puromycin (OPP) incorporation

  • Flow cytometry-based measurement of OPP intensity in specific immunophenotypic populations

• Apoptosis assessment:

  • Bone marrow cytospins for morphological assessment

  • Flow cytometry with PI and Annexin V markers

• Functional HSC assays:

  • Bone marrow transplantation experiments to assess repopulation capacity

  • Colony-forming assays to evaluate progenitor function

  • Peripheral blood count monitoring over time

• Age-dependent analysis:

  • Comparative studies between young (6-8 weeks) and older (6-7 months) mice

  • Assessment of potential recovery of specific populations with age

These methodologies have revealed that RPS12 reduction leads to decreased HSC numbers, increased apoptosis in the HSC compartment, and compromised HSC function, providing important insights into the role of ribosomal proteins in stem cell biology .

What analytical methods can detect alterations in RPS12 expression in disease states?

To assess RPS12 expression changes in pathological conditions, researchers should consider:

• Transcriptomic analysis:

  • RNA-seq to assess RPS12 mRNA expression levels

  • qRT-PCR validation of expression changes

  • Note: Increased RPS12 expression has been observed in colorectal cancers compared to matched normal colonic mucosa

• Proteomic approaches:

  • Western blot quantification using specific antibodies

  • Mass spectrometry-based quantitative proteomics

  • Immunohistochemistry for tissue-specific expression

• Experimental disease models:

  • Mouse models of RPS12 haploinsufficiency to study Diamond-Blackfan Anemia-like syndromes

  • Cancer cell line models to investigate RPS12 alterations in malignancy

• Tissue microarray analysis:

  • IHC applied to patient tissue samples

  • Validation in human colon cancer tissue has been demonstrated

• Single-cell analysis:

  • Single-cell RNA-seq to detect cell type-specific alterations

  • Single-cell proteomics for protein-level changes

These approaches can be complementary and provide multi-level evidence for RPS12 dysregulation in disease states, particularly in cancer and hematological disorders.

What is the significance of RPS12 in translational accuracy studies?

RPS12 plays a critical role in translational accuracy, making it an important subject for research in this area:

• Structural role:

  • RPS12 works with ribosomal proteins S4 and S5 to maintain translational fidelity

  • Located at a functionally important region of the ribosome

• Experimental approaches:

  • In vitro translation assays measuring miscoding rates

  • Ribosome profiling to assess translational accuracy genome-wide

  • Mutational studies of RPS12 to identify critical residues

• Evolutionary conservation:

  • High conservation across species suggests fundamental importance

  • Studies in bacterial homologs have provided insights applicable to eukaryotic systems

• Disease relevance:

  • Translational accuracy defects can contribute to protein misfolding disorders

  • RPS12 haploinsufficiency models show altered global translation patterns

• Ribosome heterogeneity:

  • RPS12 may contribute to specialized ribosomes with distinct translational properties

  • Tissue-specific effects of RPS12 depletion suggest context-dependent functions