RPL4 Antibody

What is RPL4 and what cellular functions does it perform?

RPL4 (also known as 60S ribosomal protein L4) is a critical component of the large ribosomal subunit responsible for protein synthesis in the cell. It has a calculated molecular weight of 48 kDa and is highly conserved across species . Beyond its canonical role in ribosome assembly and protein synthesis, RPL4 has several non-canonical functions including:

• Regulating the MDM2-p53 pathway, where it directly binds to MDM2 and suppresses MDM2-mediated ubiquitination and degradation of p53

• Participating in viral replication processes, notably in the Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication

• Redistributing from the cytoplasm to the nucleus during certain viral infections

What is the difference between monoclonal and polyclonal RPL4 antibodies?

The two major types of RPL4 antibodies differ in several key aspects:

What are the validated applications for RPL4 antibodies?

RPL4 antibodies have been validated for multiple research applications across different species:

How should samples be prepared for optimal RPL4 detection?

Sample preparation varies by application and experimental goals:

For Western blotting:

• Cells should be lysed in NETN buffer or similar compatible buffers

• Loading 10-50 μg of total protein per lane typically yields good results

• Heat samples at 95°C for 5 minutes in reducing SDS sample buffer

For immunohistochemistry:

• Perform heat-mediated antigen retrieval using citrate buffer pH 6.0 or TE buffer pH 9.0

• Use paraffin-embedded tissues sectioned at 4-6 μm thickness

• For plant tissues, modifications to fixation protocols may be necessary

For immunofluorescence:

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100

• Block with 1-5% BSA or serum for at least 30 minutes

How can RPL4 antibodies be used to study the MDM2-p53 regulatory pathway?

RPL4 has been identified as a novel regulator of the MDM2-p53 pathway, making RPL4 antibodies valuable tools for cancer research:

• RPL4 directly binds to MDM2 both in cells and in vitro, significantly inhibiting MDM2-mediated p53 ubiquitination and degradation

• Researchers can use co-immunoprecipitation with RPL4 antibodies to study interactions with MDM2, p53, and other ribosomal proteins such as RPL5 and RPL11

• RPL4 overexpression stabilizes p53 and activates p53 target genes including p21 and MDM2 itself

• Experimental design should include RPL4 immunoprecipitation followed by Western blotting for MDM2, p53, and other interacting partners

Methodology for studying this interaction:

• Perform co-IP using anti-RPL4 antibodies (0.5-4.0 μg for 1-3 mg of total protein lysate)

• Analyze co-precipitated proteins by Western blotting for MDM2, p53, RPL5, and RPL11

• Include appropriate controls (IgG control, input lysate)

• Consider assessing p53 stability through half-life assays following modulation of RPL4 levels

What is the role of RPL4 in viral replication and how can it be studied?

RPL4 plays a critical role in Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP), essential for EBV persistence and tumorigenesis:

• RPL4 forms a complex with EBNA1 and Nucleolin (NCL) to stabilize EBNA1 binding to oriP

• EBV infection increases RPL4 expression and redistributes it from the cytoplasm to the nucleus

• RPL4 knockdown decreases EBNA1 activation of an oriP reporter, EBNA1 DNA binding, and EBV genome numbers in lymphoblastoid cell lines

Experimental approaches:

• Use immunofluorescence with anti-RPL4 antibodies to track RPL4 relocalization during viral infection

• Employ chromatin immunoprecipitation (ChIP) with anti-RPL4 antibodies to assess binding to viral replication origins

• Combine with functional assays (reporter assays, viral genome quantification) to correlate RPL4 binding with functional outcomes

How can specificity of RPL4 antibodies be validated in experimental systems?

Validating RPL4 antibody specificity is crucial for reliable results:

• Knockdown/knockout controls: Use RPL4 shRNA knockdown or CRISPR knockout samples as negative controls

• Overexpression controls: Overexpress tagged RPL4 and confirm detection with both tag-specific and RPL4-specific antibodies

• Cross-reactivity assessment: Test in multiple species according to predicted reactivity patterns

• Multiple antibody validation: Use antibodies targeting different RPL4 epitopes and compare results

• Competitive peptide blocking: Pre-incubate antibody with immunizing peptide to confirm specificity

What are common challenges when working with RPL4 antibodies and how can they be addressed?

How should RPL4 antibodies be stored and handled to maintain optimal performance?

Based on manufacturer recommendations across multiple antibodies:

• Store concentrated antibodies at -20°C in aliquots to avoid repeated freeze-thaw cycles

• For reconstituted lyophilized antibodies, add the recommended volume of sterile water and aliquot immediately

• Most RPL4 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Spin tubes briefly before opening to collect all material that may adhere to caps or sides

• For working solutions, store at 4°C for short periods (1-2 weeks) or re-freeze in aliquots

• Optimal stability is typically one year after shipment when stored properly

How do RPL4 detection protocols differ between plant and animal systems?

RPL4 is highly conserved but requires different experimental approaches in plant versus animal systems:

For plant systems:

• Plant-specific RPL4 antibodies such as the Agrisera AS22 4787 are optimized for Arabidopsis thaliana, Horderum vulgare, and other plant species

• Expected molecular weight of plant RPL4 is typically around 26 kDa (compared to 48 kDa in mammals) due to N-terminal or C-terminal processing

• Sample preparation often requires specific grinding buffers containing PVPP to remove plant-specific secondary metabolites

• Optimal dilution for Western blotting in plant samples is typically 1:1000

For animal systems:

• Multiple mammalian-optimized antibodies show reactivity with human, mouse, and rat samples

• Standard cell lysis buffers like RIPA or NETN are typically sufficient

• Observed molecular weight is consistently around 48 kDa

How can RPL4 antibodies be used in advanced imaging techniques?

RPL4 antibodies can be employed in sophisticated imaging approaches:

• Super-resolution microscopy: RPL4 antibodies can be used to visualize ribosomal redistribution with nanometer precision

• Live-cell imaging: When coupled with cell-permeable fluorescent tags, anti-RPL4 can track ribosome dynamics

• FRET/FLIM: Can reveal proximity between RPL4 and interacting partners when using appropriate secondary antibody pairs

• Proximity ligation assay (PLA): Particularly useful for visualizing RPL4 interactions with MDM2 or viral proteins in situ

Methodological considerations for these applications include:

• Secondary antibody selection (minimal cross-reactivity, appropriate fluorophores)

• Fixation optimization (balancing antigen preservation with structural integrity)

• Appropriate controls (including competition with immunizing peptides)

• Signal amplification techniques for low-abundance interactions