RPL30 Antibody

What is RPL30 and what is its biological significance?

RPL30 is a ribosomal protein belonging to the L30E family that functions as a component of the 60S subunit of ribosomes. Ribosomes are organelles that catalyze protein synthesis, consisting of a small 40S subunit and a large 60S subunit. Together these subunits contain 4 RNA species and approximately 80 structurally distinct proteins .

RPL30 is located in the cytoplasm and is co-transcribed with the U72 small nucleolar RNA gene, which is located in its fourth intron. Like many ribosomal proteins, there are multiple processed pseudogenes of RPL30 dispersed throughout the genome .

Beyond its structural role in ribosomes, RPL30 has been shown to function as a splicing regulator. In Saccharomyces cerevisiae, RPL30 binds to purine-rich internal loops in its own pre-mRNA and mRNA to autoregulate its expression .

What are the typical applications for RPL30 antibodies in research?

RPL30 antibodies are utilized in multiple experimental applications:

RPL30 antibodies are particularly valuable in ChIP experiments, where RPL30 serves as a reliable positive control for actively transcribed genes .

What is the expected molecular weight of RPL30 in Western blot applications?

• Most commonly observed: 13 kDa

• Some antibodies report: 39 kDa

This discrepancy could be due to post-translational modifications or alternative forms of the protein. When performing Western blot analysis with RPL30 antibodies, it's important to verify which band size has been validated by the antibody manufacturer .

How should I optimize RPL30 antibody use for different applications?

For Western Blot (WB):

• Recommended dilution range: 1:500-1:2000

• Optimal protein loading: 50 μg of whole cell lysate

• Lysis buffer recommendation: NETN lysis buffer has been validated for RPL30 detection

• Detection systems: Both chemiluminescence and fluorescence detection methods are suitable

For Immunohistochemistry (IHC):

• Recommended dilution: 1:50-1:100

• Antigen retrieval: Heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol

• Detection systems: DAB (3,3'-diaminobenzidine) visualization is commonly used

For Immunofluorescence (IF):

• Recommended dilution: 1:50-1:100 or 1:100 for cultured cells

• Fixation: 4% paraformaldehyde is typically used

• Cell permeabilization: 0.1% Triton X-100 in PBS

For Chromatin Immunoprecipitation (ChIP):

• RPL30 primers are frequently used as positive controls in ChIP experiments

• The RPL30 gene is actively transcribed in all cell types and its promoter is highly enriched for histone modifications associated with active transcription

What controls should I include when using RPL30 antibodies in my experiments?

For Western Blot:

• Positive controls: Lysates from HeLa, HEK-293T, Jurkat, A549, HepG2, MCF-7, or SH-SY5Y cells all show detectable levels of RPL30

• Loading control: Housekeeping proteins such as GAPDH, β-actin, or α-tubulin

• Negative control: Use primary antibody diluent only (omit primary antibody)

For Immunohistochemistry/Immunofluorescence:

• Positive tissue controls: Pancreas and gastric tissues show reliable RPL30 expression

• Negative control: Use isotype-matched IgG at the same concentration as the RPL30 antibody

For ChIP Experiments:

• Positive control antibody: Histone H3 (D2B12) XP® Rabbit mAb can be used as a positive control for the RPL30 locus

• Negative control: Normal Rabbit IgG should show less than 0.1% enrichment of RPL30 promoter

• Expected results: Using the positive control Histone H3 antibody, RPL30 promoter enrichment should be between 2-4% of total input chromatin

How should I store and handle RPL30 antibodies to maintain their activity?

Most RPL30 antibodies share similar storage requirements:

• Long-term storage: -20°C for one year

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

• Avoid repeated freeze-thaw cycles

• Most antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• Some formulations include BSA (0.5%) for stability

Safety note: Many antibody preparations contain sodium azide as a preservative, which is a POISONOUS AND HAZARDOUS SUBSTANCE that should be handled by trained staff only .

What is the role of RPL30 in splicing regulation and how can antibodies help study this function?

RPL30 has been demonstrated to regulate its own splicing. In yeast, RPL30 binds to purine-rich internal loops in its pre-mRNA and mature mRNA to autoregulate its expression . This regulation of splicing involves:

• Binding of L30 protein to a specific site in the RPL30 transcript

• Repression of U2 snRNP recruitment

• Inhibition of the splicing process

Research has identified that position C9 in RPL30 is specifically required for L30 to repress U2 snRNP recruitment. Mutation of this site (C9U) disrupts this repression .

RPL30 antibodies can be used in the following methods to study this regulatory function:

• RNA immunoprecipitation (RIP) to capture RPL30-bound RNA species

• Chromatin immunoprecipitation (ChIP) to study the interaction of RPL30 with chromatin

• Immunoprecipitation followed by mass spectrometry to identify protein interaction partners involved in the splicing regulation

How does the cap-binding complex component Cbp80 interact with RPL30 in splicing regulation?

Research has revealed a complex relationship between Cbp80 (a component of the cap-binding complex) and RPL30 in splicing regulation:

• Deletion of Cbp80 can restore regulation of splicing in mutants with disrupted RPL30 binding sites

• Cbp80 plays distinct roles in the recognition of introns by U1 and U2 snRNP:

  • It promotes initial 5' splice site recognition by U1

  • Independently facilitates U2 recruitment depending on sequences near the 5' splice site

This interaction reveals a novel function for the cap-binding complex in splicing and demonstrates how molecular events in splicing can be targeted by regulators like RPL30 .

When studying these interactions, researchers can use:

• Co-immunoprecipitation with RPL30 antibodies to detect interaction with Cbp80

• In vitro splicing assays with recombinant L30 protein added to extracts from wild-type or cbp80Δ cells

• RNA-protein crosslinking followed by immunoprecipitation with RPL30 antibodies

Why is RPL30 commonly used as a control in ChIP experiments?

RPL30 is an ideal control for ChIP experiments for several reasons:

• Consistent expression: RPL30 is actively transcribed in virtually all cell types, making it a reliable positive control regardless of experimental system

• Chromatin signature: The RPL30 gene promoter is highly enriched for histone modifications associated with active transcription, including:

  • Histone H3 Lys4 tri-methylation

  • General histone acetylation

• Low variability: The RPL30 gene shows very low levels of histone modifications associated with heterochromatin, such as histone H3 Lys9 or Lys27 tri-methylation, making it a stable reference point

• Quantifiable enrichment: Using control histone H3 antibodies, the RPL30 promoter typically shows 2-4% enrichment relative to input chromatin, providing a reliable benchmark for successful ChIP experiments

Cell Signaling Technology provides specific RPL30 exon 3 primers (SimpleChIP® Human RPL30 Exon 3 Primers) optimized for use in SYBR® Green quantitative real-time PCR for ChIP applications .

How can I validate the specificity of my RPL30 antibody?

Multiple approaches can be used to validate RPL30 antibody specificity:

Western Blot Validation:

• Confirm a single band at the expected molecular weight (approximately 13 kDa)

• Test multiple cell lines with known RPL30 expression (e.g., HeLa, HEK-293T, Jurkat, A549)

• Include negative controls (primary antibody omission, non-specific IgG)

Protein Array Analysis:

• Some manufacturers use protein arrays containing 384 different antigens including RPL30 to analyze antibody specificity

• Based on the array interaction profile, antibodies are scored as "Supported," "Approved," or "Uncertain"

Enhanced Validation Methods:
Several enhanced validation approaches can provide stronger evidence of specificity:

• siRNA knockdown: Evaluate decrease in antibody staining intensity upon target protein downregulation

• Tagged GFP cell lines: Assess signal overlap between antibody staining and GFP-tagged protein

• Independent antibodies: Compare staining patterns of multiple antibodies directed against different epitopes on RPL30

What are common issues when using RPL30 antibodies and how can I address them?

Issue 1: Multiple bands in Western blot

• Possible causes: Non-specific binding, protein degradation, post-translational modifications

• Solutions:

  • Optimize antibody dilution (try more dilute antibody)

  • Increase blocking time or concentration

  • Use freshly prepared lysates with protease inhibitors

  • Change blocking agent (try 5% BSA instead of milk if high background)

Issue 2: Weak or no signal in IHC/IF

• Possible causes: Insufficient antigen retrieval, overfixation, low target expression

• Solutions:

  • Optimize antigen retrieval method (heat-mediated with citrate buffer pH 6 is recommended)

  • Reduce fixation time

  • Increase antibody concentration or incubation time

  • Ensure compatible detection system

Issue 3: Cross-reactivity with unexpected species

• Possible causes: Sequence homology between species

• Solutions:

  • Check sequence homology of the immunogen across species

  • Perform validation in the specific species of interest

  • Consider using antibodies raised against species-specific regions of RPL30

How can I determine if RPL30 antibodies will cross-react with samples from different species?

Cross-reactivity of RPL30 antibodies depends on sequence conservation of the immunogen region across species. Several approaches can help determine potential cross-reactivity:

• Sequence homology analysis:

  • Check the sequence homology of the immunogen region across species

  • For example, Thermo Fisher Scientific's PA5-44484 antibody targets a peptide sequence with 100% homology across multiple species (cow, dog, guinea pig, horse, human, mouse, rabbit, rat, zebrafish)

• Manufacturer's validation data:

  • Review the species reactivity information provided by manufacturers

  • For example, Boster's RPL30 antibody (A06994-1) is validated for human, monkey, mouse, and rat samples

• Published literature:

  • Search for publications that have used the antibody in your species of interest

  • Contact the authors for specific validation protocols

• Experimental validation:

  • Test the antibody on positive control samples from your species of interest

  • Compare with known positive controls from validated species

Data on reported cross-reactivity:
The following table summarizes reported cross-reactivity for selected RPL30 antibodies:

How is RPL30 being used as a marker in current research studies?

RPL30 serves several important functions in current research:

Housekeeping Gene Control:

• As a ribosomal protein, RPL30 is often used as a housekeeping gene control in gene expression studies

• It's particularly valuable in ChIP experiments as a positive control for actively transcribed genes

Splicing Regulation Studies:

• The RPL30 system is used as a model to study autoregulation of splicing and RNA-protein interactions

• RPL30 mutations that affect splicing regulation (e.g., C9U mutation) are valuable tools for investigating splicing mechanisms

Ribosome Biogenesis Research:

• RPL30 antibodies are used to study the assembly and function of ribosomes

• They help investigate how defects in ribosomal proteins contribute to diseases known as ribosomopathies

Novel RNA-Affinity Proteogenomics:

• RPL30 has been included in emerging approaches that combine RNA-affinity purification with proteomics to dissect tumor heterogeneity

• This approach helps identify personalized markers for precision prognosis of cancer

What is the significance of the interaction between RPL30 and U72 small nucleolar RNA?

The RPL30 gene contains the U72 small nucleolar RNA gene within its fourth intron . This genomic arrangement has several important implications:

• Co-regulation: The transcription of RPL30 and U72 snoRNA are linked, suggesting coordinated function

• Evolutionary conservation: This arrangement is conserved across species, indicating functional importance

• Ribosome biogenesis connection: U72 is likely involved in ribosomal RNA processing, creating a functional link between RPL30 (a ribosomal protein) and U72 (involved in ribosome assembly)

• Splicing regulation: The presence of U72 within the RPL30 gene may play a role in the regulation of RPL30 splicing, potentially contributing to the autoregulatory mechanism

Research into this interaction typically employs:

• RNA immunoprecipitation with RPL30 antibodies to capture associated RNAs

• RNA-seq to identify the full complement of RNAs associated with RPL30

• Deletion analysis to determine the functional significance of the U72 snoRNA in RPL30 expression

While this area remains under investigation, the intronic location of U72 within RPL30 represents an interesting example of genomic organization that likely reflects functional coordination between ribosomal proteins and the small RNAs involved in ribosome assembly.

RPL30 Antibody Researcher FAQs: Scientific Applications, Methods, and Validation

RPL30 (Ribosomal Protein L30) is a component of the 60S ribosomal subunit involved in protein synthesis. The following frequently asked questions address common research scenarios, experimental approaches, and troubleshooting strategies when working with RPL30 antibodies.

What is RPL30 and what is its biological significance?

RPL30 is a ribosomal protein belonging to the L30E family that functions as a component of the 60S subunit of ribosomes. Ribosomes are organelles that catalyze protein synthesis, consisting of a small 40S subunit and a large 60S subunit. Together these subunits contain 4 RNA species and approximately 80 structurally distinct proteins .

RPL30 is located in the cytoplasm and is co-transcribed with the U72 small nucleolar RNA gene, which is located in its fourth intron. Like many ribosomal proteins, there are multiple processed pseudogenes of RPL30 dispersed throughout the genome .

Beyond its structural role in ribosomes, RPL30 has been shown to function as a splicing regulator. In Saccharomyces cerevisiae, RPL30 binds to purine-rich internal loops in its own pre-mRNA and mRNA to autoregulate its expression .

What are the typical applications for RPL30 antibodies in research?

RPL30 antibodies are utilized in multiple experimental applications:

RPL30 antibodies are particularly valuable in ChIP experiments, where RPL30 serves as a reliable positive control for actively transcribed genes .

What is the expected molecular weight of RPL30 in Western blot applications?

• Most commonly observed: 13 kDa

• Some antibodies report: 39 kDa

This discrepancy could be due to post-translational modifications or alternative forms of the protein. When performing Western blot analysis with RPL30 antibodies, it's important to verify which band size has been validated by the antibody manufacturer .

How should I optimize RPL30 antibody use for different applications?

For Western Blot (WB):

• Recommended dilution range: 1:500-1:2000

• Optimal protein loading: 50 μg of whole cell lysate

• Lysis buffer recommendation: NETN lysis buffer has been validated for RPL30 detection

• Detection systems: Both chemiluminescence and fluorescence detection methods are suitable

For Immunohistochemistry (IHC):

• Recommended dilution: 1:50-1:100

• Antigen retrieval: Heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol

• Detection systems: DAB (3,3'-diaminobenzidine) visualization is commonly used

For Immunofluorescence (IF):

• Recommended dilution: 1:50-1:100 or 1:100 for cultured cells

• Fixation: 4% paraformaldehyde is typically used

• Cell permeabilization: 0.1% Triton X-100 in PBS

For Chromatin Immunoprecipitation (ChIP):

• RPL30 primers are frequently used as positive controls in ChIP experiments

• The RPL30 gene is actively transcribed in all cell types and its promoter is highly enriched for histone modifications associated with active transcription

What controls should I include when using RPL30 antibodies in my experiments?

For Western Blot:

• Positive controls: Lysates from HeLa, HEK-293T, Jurkat, A549, HepG2, MCF-7, or SH-SY5Y cells all show detectable levels of RPL30

• Loading control: Housekeeping proteins such as GAPDH, β-actin, or α-tubulin

• Negative control: Use primary antibody diluent only (omit primary antibody)

For Immunohistochemistry/Immunofluorescence:

• Positive tissue controls: Pancreas and gastric tissues show reliable RPL30 expression

• Negative control: Use isotype-matched IgG at the same concentration as the RPL30 antibody

For ChIP Experiments:

• Positive control antibody: Histone H3 (D2B12) XP® Rabbit mAb can be used as a positive control for the RPL30 locus

• Negative control: Normal Rabbit IgG should show less than 0.1% enrichment of RPL30 promoter

• Expected results: Using the positive control Histone H3 antibody, RPL30 promoter enrichment should be between 2-4% of total input chromatin

How should I store and handle RPL30 antibodies to maintain their activity?

Most RPL30 antibodies share similar storage requirements:

• Long-term storage: -20°C for one year

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

• Avoid repeated freeze-thaw cycles

• Most antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• Some formulations include BSA (0.5%) for stability

Safety note: Many antibody preparations contain sodium azide as a preservative, which is a POISONOUS AND HAZARDOUS SUBSTANCE that should be handled by trained staff only .

What is the role of RPL30 in splicing regulation and how can antibodies help study this function?

RPL30 has been demonstrated to regulate its own splicing. In yeast, RPL30 binds to purine-rich internal loops in its pre-mRNA and mature mRNA to autoregulate its expression . This regulation of splicing involves:

• Binding of L30 protein to a specific site in the RPL30 transcript

• Repression of U2 snRNP recruitment

• Inhibition of the splicing process

Research has identified that position C9 in RPL30 is specifically required for L30 to repress U2 snRNP recruitment. Mutation of this site (C9U) disrupts this repression .

RPL30 antibodies can be used in the following methods to study this regulatory function:

• RNA immunoprecipitation (RIP) to capture RPL30-bound RNA species

• Chromatin immunoprecipitation (ChIP) to study the interaction of RPL30 with chromatin

• Immunoprecipitation followed by mass spectrometry to identify protein interaction partners involved in the splicing regulation

How does the cap-binding complex component Cbp80 interact with RPL30 in splicing regulation?

Research has revealed a complex relationship between Cbp80 (a component of the cap-binding complex) and RPL30 in splicing regulation:

• Deletion of Cbp80 can restore regulation of splicing in mutants with disrupted RPL30 binding sites

• Cbp80 plays distinct roles in the recognition of introns by U1 and U2 snRNP:

  • It promotes initial 5' splice site recognition by U1

  • Independently facilitates U2 recruitment depending on sequences near the 5' splice site

This interaction reveals a novel function for the cap-binding complex in splicing and demonstrates how molecular events in splicing can be targeted by regulators like RPL30 .

When studying these interactions, researchers can use:

• Co-immunoprecipitation with RPL30 antibodies to detect interaction with Cbp80

• In vitro splicing assays with recombinant L30 protein added to extracts from wild-type or cbp80Δ cells

• RNA-protein crosslinking followed by immunoprecipitation with RPL30 antibodies

Why is RPL30 commonly used as a control in ChIP experiments?

RPL30 is an ideal control for ChIP experiments for several reasons:

• Consistent expression: RPL30 is actively transcribed in virtually all cell types, making it a reliable positive control regardless of experimental system

• Chromatin signature: The RPL30 gene promoter is highly enriched for histone modifications associated with active transcription, including:

  • Histone H3 Lys4 tri-methylation

  • General histone acetylation

• Low variability: The RPL30 gene shows very low levels of histone modifications associated with heterochromatin, such as histone H3 Lys9 or Lys27 tri-methylation, making it a stable reference point

• Quantifiable enrichment: Using control histone H3 antibodies, the RPL30 promoter typically shows 2-4% enrichment relative to input chromatin, providing a reliable benchmark for successful ChIP experiments

Cell Signaling Technology provides specific RPL30 exon 3 primers (SimpleChIP® Human RPL30 Exon 3 Primers) optimized for use in SYBR® Green quantitative real-time PCR for ChIP applications .

How can I validate the specificity of my RPL30 antibody?

Multiple approaches can be used to validate RPL30 antibody specificity:

Western Blot Validation:

• Confirm a single band at the expected molecular weight (approximately 13 kDa)

• Test multiple cell lines with known RPL30 expression (e.g., HeLa, HEK-293T, Jurkat, A549)

• Include negative controls (primary antibody omission, non-specific IgG)

Protein Array Analysis:

• Some manufacturers use protein arrays containing 384 different antigens including RPL30 to analyze antibody specificity

• Based on the array interaction profile, antibodies are scored as "Supported," "Approved," or "Uncertain"

Enhanced Validation Methods:
Several enhanced validation approaches can provide stronger evidence of specificity:

• siRNA knockdown: Evaluate decrease in antibody staining intensity upon target protein downregulation

• Tagged GFP cell lines: Assess signal overlap between antibody staining and GFP-tagged protein

• Independent antibodies: Compare staining patterns of multiple antibodies directed against different epitopes on RPL30

What are common issues when using RPL30 antibodies and how can I address them?

Issue 1: Multiple bands in Western blot

• Possible causes: Non-specific binding, protein degradation, post-translational modifications

• Solutions:

  • Optimize antibody dilution (try more dilute antibody)

  • Increase blocking time or concentration

  • Use freshly prepared lysates with protease inhibitors

  • Change blocking agent (try 5% BSA instead of milk if high background)

Issue 2: Weak or no signal in IHC/IF

• Possible causes: Insufficient antigen retrieval, overfixation, low target expression

• Solutions:

  • Optimize antigen retrieval method (heat-mediated with citrate buffer pH 6 is recommended)

  • Reduce fixation time

  • Increase antibody concentration or incubation time

  • Ensure compatible detection system

Issue 3: Cross-reactivity with unexpected species

• Possible causes: Sequence homology between species

• Solutions:

  • Check sequence homology of the immunogen across species

  • Perform validation in the specific species of interest

  • Consider using antibodies raised against species-specific regions of RPL30

How can I determine if RPL30 antibodies will cross-react with samples from different species?

Cross-reactivity of RPL30 antibodies depends on sequence conservation of the immunogen region across species. Several approaches can help determine potential cross-reactivity:

• Sequence homology analysis:

  • Check the sequence homology of the immunogen region across species

  • For example, Thermo Fisher Scientific's PA5-44484 antibody targets a peptide sequence with 100% homology across multiple species (cow, dog, guinea pig, horse, human, mouse, rabbit, rat, zebrafish)

• Manufacturer's validation data:

  • Review the species reactivity information provided by manufacturers

  • For example, Boster's RPL30 antibody (A06994-1) is validated for human, monkey, mouse, and rat samples

• Published literature:

  • Search for publications that have used the antibody in your species of interest

  • Contact the authors for specific validation protocols

• Experimental validation:

  • Test the antibody on positive control samples from your species of interest

  • Compare with known positive controls from validated species

Data on reported cross-reactivity:
The following table summarizes reported cross-reactivity for selected RPL30 antibodies:

How is RPL30 being used as a marker in current research studies?

RPL30 serves several important functions in current research:

Housekeeping Gene Control:

• As a ribosomal protein, RPL30 is often used as a housekeeping gene control in gene expression studies

• It's particularly valuable in ChIP experiments as a positive control for actively transcribed genes

Splicing Regulation Studies:

• The RPL30 system is used as a model to study autoregulation of splicing and RNA-protein interactions

• RPL30 mutations that affect splicing regulation (e.g., C9U mutation) are valuable tools for investigating splicing mechanisms

Ribosome Biogenesis Research:

• RPL30 antibodies are used to study the assembly and function of ribosomes

• They help investigate how defects in ribosomal proteins contribute to diseases known as ribosomopathies

Novel RNA-Affinity Proteogenomics:

• RPL30 has been included in emerging approaches that combine RNA-affinity purification with proteomics to dissect tumor heterogeneity

• This approach helps identify personalized markers for precision prognosis of cancer

What is the significance of the interaction between RPL30 and U72 small nucleolar RNA?

The RPL30 gene contains the U72 small nucleolar RNA gene within its fourth intron . This genomic arrangement has several important implications:

• Co-regulation: The transcription of RPL30 and U72 snoRNA are linked, suggesting coordinated function

• Evolutionary conservation: This arrangement is conserved across species, indicating functional importance

• Ribosome biogenesis connection: U72 is likely involved in ribosomal RNA processing, creating a functional link between RPL30 (a ribosomal protein) and U72 (involved in ribosome assembly)

• Splicing regulation: The presence of U72 within the RPL30 gene may play a role in the regulation of RPL30 splicing, potentially contributing to the autoregulatory mechanism

Research into this interaction typically employs:

• RNA immunoprecipitation with RPL30 antibodies to capture associated RNAs

• RNA-seq to identify the full complement of RNAs associated with RPL30

• Deletion analysis to determine the functional significance of the U72 snoRNA in RPL30 expression

While this area remains under investigation, the intronic location of U72 within RPL30 represents an interesting example of genomic organization that likely reflects functional coordination between ribosomal proteins and the small RNAs involved in ribosome assembly.