PRDM11 Antibody

What is PRDM11 and what are its known functions?

PRDM11 (PR/SET Domain 11) is a protein-coding gene that belongs to the PR-domain family of transcriptional regulators. Gene Ontology (GO) annotations for PRDM11 include nucleic acid binding and methyltransferase activity . PRDM11 functions primarily as a transcriptional regulator and has been characterized as a putative tumor suppressor that controls the expression of key oncogenes .

Functionally, PRDM11 has been shown to:

• Associate with transcriptional start sites of target genes

• Regulate important oncogenes such as FOS and JUN

• Inhibit cell proliferation when overexpressed

• Induce apoptosis when overexpressed

The protein is localized in both the cytoplasm and nucleus, suggesting multiple functional roles depending on cellular context .

While the calculated molecular weight of PRDM11 is approximately 53-57 kDa, researchers often observe bands at approximately 58 kDa in Western blot applications . This discrepancy between calculated and observed molecular weights is common for many proteins and can be attributed to post-translational modifications, protein folding, or other factors affecting electrophoretic mobility.

As noted in the product information: "The actual band is not consistent with the expectation. Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

How can PRDM11 antibodies be used to study its role in lymphomagenesis?

PRDM11 has been identified as a tumor suppressor in lymphoma development, particularly in MYC-driven lymphomagenesis . Researchers can utilize PRDM11 antibodies to investigate this relationship through several experimental approaches:

Research has demonstrated that genome-wide mapping of PRDM11 binding sites coupled with transcriptome sequencing in human DLBCL cells showed that PRDM11 associates with transcriptional start sites of target genes and regulates important oncogenes .

What are the methodological considerations when using PRDM11 antibodies in chromatin studies?

When implementing PRDM11 antibodies for chromatin-related research, consider the following methodological guidelines:

• Cross-linking optimization: Since PRDM11 functions as a transcriptional regulator that associates with transcriptional start sites , optimal cross-linking conditions are crucial for ChIP experiments. Generally, 1% formaldehyde for 10 minutes at room temperature works well for most transcription factors.

• Sonication parameters: Aim for chromatin fragments between 200-500 bp for optimal resolution of binding sites.

• Antibody validation: Confirm specificity through knockdown/knockout controls before proceeding with genome-wide studies.

• Sequential ChIP: Consider sequential ChIP (ChIP-reChIP) to study co-occupancy with other transcription factors or histone marks, particularly those related to transcriptional regulation.

• Integration with transcriptomic data: As demonstrated in previous research , combining ChIP-seq data with RNA-seq can provide insights into PRDM11's regulatory impact on gene expression.

Based on the literature, PRDM11 primarily regulates oncogenes such as FOS and JUN, suggesting a focus on these loci when designing validation experiments .

How is PRDM11 expression altered in cancer, and how can antibodies help investigate this?

PRDM11 expression alterations have been documented in several cancer types:

Antibody-based approaches to investigate these alterations include:

• Immunohistochemistry (IHC): Using PRDM11 antibodies at dilutions of 1:200-1:500 to assess expression patterns in tissue sections.

• Tissue microarrays: For high-throughput analysis of PRDM11 expression across multiple patient samples.

• Multiplexed immunofluorescence: To correlate PRDM11 expression with other markers of cancer progression.

• Western blotting: To quantify PRDM11 protein levels in cancer cell lines or patient-derived samples.

Researchers should note that alterations in PRDM11 may occur at both the expression level and through mutations affecting protein function, necessitating a comprehensive approach combining antibody-based protein detection with genetic analysis.

What are the optimal protocols for Western blotting with PRDM11 antibodies?

For optimal Western blot results with PRDM11 antibodies, follow these evidence-based recommendations:

• Sample preparation:

  • Use RIPA buffer containing protease inhibitors for cell lysis

  • Load 20-40 μg of total protein per lane

• Gel electrophoresis:

  • Use 10% SDS-PAGE gels for optimal resolution of PRDM11 (~58 kDa observed molecular weight)

• Transfer conditions:

  • Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

• Blocking:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute PRDM11 antibody 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

• Detection:

  • Apply appropriate secondary antibody (anti-rabbit IgG for most PRDM11 antibodies)

  • Develop using enhanced chemiluminescence

• Expected results:

  • Anticipate detection at approximately 58 kDa, which may differ from the calculated molecular weight (53-57 kDa)

It's important to note that researchers may observe multiple bands, as mentioned in product documentation: "If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane."

What validation steps should be performed for new PRDM11 antibodies?

Thorough validation of PRDM11 antibodies is essential for reliable experimental results:

• Specificity validation:

  • Positive control: Express recombinant PRDM11 in a model system

  • Negative control: Use PRDM11 knockout/knockdown samples

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide

• Application-specific validation:

  • For Western blotting: Verify single band at expected molecular weight (~58 kDa)

  • For IHC: Compare staining pattern with published results and RNA expression data

  • For immunoprecipitation: Confirm enrichment of PRDM11 by mass spectrometry

• Cross-reactivity assessment:

  • Test antibody against related PRDM family members

  • Evaluate in multiple species if cross-reactivity is claimed

• Reproducibility testing:

  • Test antibody across different lots

  • Assess consistency across multiple biological replicates

• Dilution optimization:

  • Perform dilution series to determine optimal concentration

  • For Western blot: Test 1:500-1:2000 range

  • For IHC: Test 1:200-1:500 range

Remember that antibodies are highly specific research tools that require proper validation to ensure experimental reliability and reproducibility.

How should PRDM11 antibodies be stored to maintain optimal activity?

Proper storage of PRDM11 antibodies is crucial for maintaining their activity and specificity:

• Temperature conditions:

  • Store at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles which can degrade antibody performance

• Aliquoting recommendations:

  • Upon receipt, prepare small working aliquots (10-20 μL)

  • Thaw only the required amount for each experiment

• Buffer considerations:

  • PRDM11 antibodies are typically supplied in phosphate buffered solution, pH 7.4, containing 0.05% stabilizer and 50% glycerol

  • Do not dilute the stock solution unless immediately using

• Handling during use:

  • Keep on ice when in use

  • Return to -20°C promptly after use

• Shipping considerations:

  • Products are typically shipped with ice packs

  • Upon receipt, store immediately at the recommended temperature

Following these storage guidelines will help ensure consistent performance of PRDM11 antibodies throughout your research project.

Why might Western blotting with PRDM11 antibodies show inconsistent results?

Inconsistent results when using PRDM11 antibodies in Western blotting can stem from several factors:

• Inconsistent molecular weight detection:

  • The observed molecular weight (58 kDa) often differs from calculated values (53-57 kDa)

  • This discrepancy is attributed to post-translational modifications and protein conformation

• Multiple bands:

  • May indicate different protein isoforms or post-translational modifications

  • "If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane"

• Technical variables affecting reproducibility:

  • Incomplete transfer: Optimize transfer time and voltage

  • Inefficient blocking: Increase blocking time or try alternative blocking agents

  • Secondary antibody cross-reactivity: Use highly cross-adsorbed secondary antibodies

• Sample preparation issues:

  • Protein degradation: Ensure complete protease inhibition

  • Insufficient denaturation: Verify heating time and temperature with sample buffer

• Antibody-specific considerations:

  • Optimal dilution may vary by lot: Test dilution series with each new lot

  • Storage conditions: Avoid freeze-thaw cycles that may reduce antibody activity

When troubleshooting, systematically address each variable while maintaining detailed records of experimental conditions to identify the source of inconsistency.

What are the common pitfalls when using PRDM11 antibodies in immunohistochemistry?

Researchers may encounter several challenges when implementing PRDM11 antibodies in immunohistochemistry:

• Fixation-dependent epitope masking:

  • Excessive fixation can mask PRDM11 epitopes

  • Solution: Optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 is often effective)

• Nonspecific background staining:

  • May result from insufficient blocking or high antibody concentration

  • Solution: Extend blocking time to 1-2 hours and optimize antibody dilution (1:200-1:500 recommended)

• Variable subcellular localization:

  • PRDM11 is reported in both cytoplasm and nucleus

  • Solution: Document and analyze both locations separately, as differential localization may have biological significance

• Tissue-specific expression differences:

  • Expression patterns may vary across tissue types

  • Solution: Include positive control tissues with known PRDM11 expression

• Interpretation challenges:

  • Distinguishing specific staining from background

  • Solution: Always include appropriate negative controls (omitting primary antibody or using isotype control)

• Reproducibility issues:

  • Batch-to-batch variation in antibody performance

  • Solution: Maintain consistent lot usage throughout a study or revalidate each new lot

Following immunogen sequence information provided in product documentation can help evaluate potential cross-reactivity with other epitopes in the tissue of interest .

How can researchers distinguish between specific and non-specific signals when using PRDM11 antibodies?

Distinguishing specific from non-specific signals is critical for accurate interpretation of PRDM11 antibody results:

• Essential controls for validation:

  • Positive control: Tissue or cell line with confirmed PRDM11 expression

  • Negative control: PRDM11 knockout/knockdown samples

  • Technical negative: Primary antibody omission

  • Blocking peptide competition: Pre-incubate antibody with immunizing peptide

• Molecular weight verification in Western blotting:

  • Specific signal should appear at the expected molecular weight (~58 kDa)

  • Non-specific bands often appear at drastically different molecular weights

• Signal pattern analysis in immunostaining:

  • Specific staining follows expected subcellular localization (cytoplasm and nucleus for PRDM11)

  • Non-specific staining often appears diffuse or in unexpected cellular compartments

• Correlation with orthogonal methods:

  • Compare protein detection with mRNA expression data

  • Verify with multiple antibodies targeting different epitopes of PRDM11

• Concentration-dependent signal assessment:

  • Specific signals typically show dose-dependent intensity with antibody dilution

  • Non-specific background often persists across multiple dilutions

The immunogen sequence for PRDM11 antibodies (e.g., "QVDFWFCESCQEYFVDECPNHGPPVFVSDTPVPVGIPDRAALTIPQGMEVVKDTSGESDVRCVNEVIPKGHIFGPYEGQISTQD" ) can be used to predict potential cross-reactivity through sequence alignment tools.

How can PRDM11 antibodies be used to study its role in tumor suppression?

PRDM11 has been characterized as a putative tumor suppressor , and antibodies can be strategically employed to investigate this function:

This comprehensive approach can help elucidate how PRDM11 exerts its tumor suppressive functions and potentially identify therapeutic opportunities.

What are the considerations for using PRDM11 antibodies in single-cell analysis techniques?

As single-cell technologies gain prominence in research, several considerations apply when incorporating PRDM11 antibodies:

• Antibody validation for single-cell applications:

  • Verify specificity at low protein concentrations typical of single cells

  • Test for minimal background in flow cytometry or mass cytometry applications

  • Optimize fixation and permeabilization protocols for intracellular detection

• Integration with single-cell genomic data:

  • PRDM11 antibodies can complement single-cell proteo-genomic reference maps

  • Consider using PRDM11 antibodies alongside other markers to identify specific cell populations

• Technical considerations for single-cell proteomics:

  • Signal amplification may be necessary due to low protein abundance

  • Careful titration to determine optimal antibody concentration

  • Use fluorophores with minimal spectral overlap for multiplexed detection

• Application-specific adjustments:

  • For flow cytometry: Consider using brightness-optimized fluorophore conjugates

  • For imaging mass cytometry: Select metal tags with high detection sensitivity

  • For CyTOF: Validate metal-conjugated antibodies specifically

• Data integration strategies:

  • Develop computational approaches to correlate PRDM11 protein levels with transcriptomic data

  • Consider reference mapping as described in high-content single-cell proteo-genomic studies

These considerations will help researchers effectively incorporate PRDM11 antibodies into emerging single-cell analysis pipelines.

How can researchers study the methyltransferase activity of PRDM11 using antibodies?

PRDM11 possesses methyltransferase activity , which can be investigated using specialized approaches involving antibodies:

• Activity-correlated expression analysis:

  • Use PRDM11 antibodies alongside histone modification antibodies

  • Correlate PRDM11 expression with specific histone marks using dual immunofluorescence

• ChIP-seq for methylation targets:

  • Perform sequential ChIP (ChIP-reChIP) with PRDM11 antibodies followed by antibodies against specific histone modifications

  • Map co-occurrence of PRDM11 binding and methylation marks genome-wide

• In vitro methyltransferase assays:

  • Immunoprecipitate PRDM11 using specific antibodies

  • Test methyltransferase activity of the immunoprecipitated protein on histone substrates

  • Detect methylation using modification-specific antibodies

• Domain-specific antibody approaches:

  • Utilize antibodies targeting different domains of PRDM11 to determine structure-function relationships

  • Particularly focus on the PR/SET domain responsible for methyltransferase activity

• Protein complex identification:

  • Use PRDM11 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Identify interaction partners that might regulate methyltransferase activity

This methodological framework will help researchers investigate the enzymatic functions of PRDM11 in relation to its role in transcriptional regulation and tumor suppression.