RPL22 Antibody

What is RPL22 and why is it important in research?

RPL22 (also known as EAP, HBP15/L22, and 60S ribosomal protein L22) is a 14.8 kDa protein that functions as a component of the large ribosomal subunit. Beyond its canonical role in protein synthesis, RPL22 has been implicated in T-cell development, cancer progression, and regulation of its paralog RPL22L1 . The protein is particularly significant because it demonstrates both ribosomal and extraribosomal functions, including direct binding to specific RNA sequences and involvement in pre-mRNA splicing .

What types of RPL22 antibodies are available and what applications are they validated for?

Multiple suppliers offer anti-RPL22 antibodies with various applications and species reactivity:

Most antibodies are validated for Western blotting (1:500-1:1000 dilution), with some also suitable for immunoprecipitation, immunofluorescence, and immunohistochemistry applications .

How should I optimize Western blotting conditions for RPL22 detection?

For optimal Western blot detection of RPL22:

• Use 15% SDS-PAGE gels due to RPL22's small size (14.8-15 kDa)

• Load adequate protein (20-30 μg of total protein lysate)

• Transfer to PVDF membrane using standard protocols

• Block with 5% non-fat milk or BSA in TBST

• Incubate with primary antibodies at recommended dilutions (typically 1:500-1:1000)

• For visualization, ECL technique works effectively with most anti-RPL22 antibodies

When troubleshooting, be aware that RPL22 may appear between 15-18 kDa on Western blots depending on post-translational modifications .

What are the best practices for immunoprecipitation of RPL22-containing complexes?

For effective immunoprecipitation of RPL22 complexes:

• Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate

• Include RNase inhibitors (RNasin) and heparin if studying RPL22-RNA interactions

• For polysome immunoprecipitation, use high-salt buffer washes to reduce non-specific binding

• When assessing ribosome association, include magnesium in buffers to maintain ribosomal integrity

• For cell-type specific studies, consider the RiboTag approach with HA-tagged RPL22

This approach has been validated for isolating intact RNA with RNA integrity number (RIN) values between 8.0-9.2, suitable for downstream applications including RNA-seq and qRT-PCR .

How can I implement the RiboTag methodology using RPL22 antibodies for cell-type specific translatome analysis?

The RiboTag methodology enables cell-type specific isolation of actively translating mRNAs by:

• Genetic preparation: Use mice with a modified Rpl22 allele (Rpl22-HA) that expresses HA-tagged RPL22 upon Cre recombination

• For non-genetic models: Implement AAV-DIO-Rpl22-HA viral delivery to target cells expressing Cre recombinase

• Tissue processing:

  • Homogenize tissue in polysome buffer containing cycloheximide, heparin, and RNase inhibitors

  • Clear homogenates by centrifugation (10,000 g for 10 minutes)

• Immunoprecipitation:

  • Incubate cleared lysates with anti-HA antibody-coupled magnetic beads overnight at 4°C

  • Wash with high-salt buffer to remove non-specific binding

• RNA extraction and analysis:

  • Extract RNA using standard methods

  • Verify RNA integrity (RIN values >8 indicate good quality)

  • Proceed with RNA-seq, microarray, or qRT-PCR analysis

This approach has been validated in various tissues including brain, testis, and tumor samples from mouse models .

How can I distinguish between RPL22 and its paralog RPL22L1 in experimental settings?

Distinguishing between RPL22 and RPL22L1 requires careful experimental design:

• Antibody selection: Use paralog-specific antibodies such as RPL22L1 (E9P6N) Rabbit mAb for specific detection of RPL22L1

• Mass spectrometry approach:

  • Employ multiple reaction monitoring (MRM) targeting unique peptides

  • Use at least 3-4 peptide targets for each protein with 6-8 fragment ions per peptide

  • Sum integrated MRM peak areas for all transitions to quantify relative amounts

• RT-PCR analysis:

  • Design primers specific to unique regions in each paralog

  • For RPL22L1, avoid exon 2 which contains a conserved Rpl22 RNA-binding motif

• Functional validation:

  • Knockout/knockdown studies targeting each paralog individually

  • Rescue experiments to confirm specificity

Research shows that RPL22 and RPL22L1 have antagonistic functions in some contexts, with RPL22 directly repressing RPL22L1 expression .

What are common issues in RPL22 antibody-based experiments and how can they be addressed?

When interpreting results, remember that RPL22 expression may vary significantly across tissues, with notable expression in lymphoid tissues and variable expression in cancer cell lines .

How should I interpret RPL22 antibody data in the context of cancer research?

When studying RPL22 in cancer contexts:

• Mutation status assessment:

  • Monoallelic inactivation of RPL22 is observed in ~10% of T-ALL patients and correlates with poor survival

  • Biallelic mutations are more common in solid tumors than hematological malignancies

• Expression pattern analysis:

  • Compare expression across multiple cell types within the tumor microenvironment

  • Consider using RiboTag methodology for cell-type specific analysis in heterogeneous tumor samples

• Functional interpretation:

  • In lymphoma models, Rpl22+/- accelerates disease progression through NF-κB activation and Lin28B induction

  • Paradoxically, Rpl22-/- may restrict lymphoma dissemination while promoting primary tumor growth and vascularization

• Alternative splicing assessment:

  • Examine potential effects on pre-mRNA splicing, particularly of genes involved in TGF-β signaling

How can I implement multi-cell type ribosomal tagging for simultaneous translatome analysis?

Recent advances allow simultaneous translatome analysis from multiple cell types within the same sample:

• Dual-tagging strategy:

  • Utilize Cdh5-CRE-ERT2/RiboTag+/+ mouse model expressing HA-tagged RPL22 in endothelial cells

  • Introduce FLAG-tagged RPL22 in other cell populations via lentiviral vectors

  • Select cells with stable expression using puromycin

• Sample processing:

  • Pulverize tissue in liquid nitrogen

  • Homogenize in lysis buffer using Dounce homogenizers

  • Perform sequential immunoprecipitations with anti-HA and anti-FLAG antibodies

• Validation:

  • Confirm tag expression in distinct cell populations via immunofluorescence

  • Use CD31 co-staining to verify endothelial-specific expression of HA-tagged RPL22

  • Check RNA quality (RIN scores) before proceeding to sequencing

This approach has been successfully implemented to simultaneously isolate high-quality transcripts from endothelial cells and tumor cells within intact melanomas .

What are emerging research directions for RPL22 antibodies beyond canonical ribosomal functions?

Emerging research areas include:

• Nuclear functions:

  • Investigating RPL22's role in pre-mRNA splicing

  • Studying developmental regulation of nuclear retention of RPL22

  • Examining direct binding of RPL22 to RNA motifs in pre-mRNAs

• Cancer biology applications:

  • Investigating RPL22 as a tumor suppressor in T-ALL and other malignancies

  • Exploring RPL22's role in tumor cell migration and metastasis

  • Examining RPL22/RPL22L1 balance in cancer development

• Developmental biology:

  • Studying RPL22's role in Smad2-dependent TGF-β signaling

  • Investigating morphogenesis control via RPL22/RPL22L1 balance

• Therapeutic target validation:

  • Using RPL22 antibodies to validate potential therapeutic interventions targeting ribosome heterogeneity

  • Exploring synthetic lethality approaches based on RPL22 status in cancer cells