PRDM11 Antibody, HRP conjugated

What is PRDM11 and why is it significant in research?

PRDM11 (PR/SET Domain 11) is a protein-coding gene with significant roles in transcription regulation. Gene Ontology (GO) annotations indicate it possesses nucleic acid binding and methyltransferase activity capabilities . PRDM11 is found in both cytoplasmic and nuclear cellular compartments, suggesting multifunctional roles in cellular processes . Research interest in PRDM11 has increased due to its association with Hermansky-Pudlak Syndrome 1, making reliable antibody detection crucial for understanding its pathological mechanisms .

What are the primary applications for PRDM11 antibodies in research?

PRDM11 antibodies are predominantly utilized in Western Blotting (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), and Immunofluorescence (IF) applications . HRP-conjugated variants specifically eliminate the need for secondary antibody incubation steps, providing more direct detection capabilities in enzyme-linked immunoassays and Western blot applications. The specific reactivity of commercially available antibodies varies, with some demonstrating reactivity only with human PRDM11 while others cross-react with mouse, cow, horse, and other mammalian PRDM11 proteins .

How does PRDM11 compare functionally with other PRDM family members?

While limited direct comparative data exists in the search results, it's notable that PRDM11 shares functional domains with other PRDM family members. PRDM7 is identified as an important paralog . Unlike the well-studied PRDM1, which functions as a master regulator in T cell hyporesponsiveness and B cell differentiation, PRDM11's exact regulatory network remains less characterized . The PR/SET domain common to this family suggests potential histone methyltransferase activity, though specific enzymatic functions of PRDM11 require further investigation.

What is the optimal protocol for Western blotting using HRP-conjugated PRDM11 antibodies?

For optimal Western blot results with HRP-conjugated PRDM11 antibodies, researchers should implement the following protocol:

• Sample preparation: Extract proteins from target tissues (verified samples include mouse heart and lung for certain antibodies)

• SDS-PAGE separation: Load equivalent protein amounts (typically 20-50μg) per lane

• Transfer: Use PVDF or nitrocellulose membranes for optimal protein binding

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Apply HRP-conjugated PRDM11 antibody at appropriate dilution (typically 1:500-1:2000)

• Washing: Perform 3-5 washes with TBST buffer

• Detection: Directly apply chemiluminescent substrate since secondary antibody is unnecessary with HRP-conjugated antibodies

• Analysis: Expected molecular weight observation may vary from calculated weight (calculated: 53-57 kDa; observed: approximately 58 kDa)

Importantly, researchers should note that observed molecular weight may not match expected weight due to post-translational modifications or other factors affecting protein mobility .

What control samples are recommended for validating PRDM11 antibody specificity?

To establish antibody specificity, researchers should implement multiple controls:

• Positive tissue controls: Mouse heart and mouse lung tissues have been validated for certain PRDM11 antibodies

• Negative controls: Use samples known to lack PRDM11 expression

• Blocking peptide competition: Pre-incubate antibody with immunizing peptide/protein (e.g., recombinant human PRDM11)

• Knockdown validation: Compare signals between wild-type cells and PRDM11 knockdown cells

• Epitope specificity verification: When using antibodies targeting specific regions (e.g., AA 63-215 or C-terminus), confirm region-specific binding patterns

This multi-layered validation approach ensures signal specificity and minimizes false positive results in experimental applications.

How should researchers optimize ELISA protocols using HRP-conjugated PRDM11 antibodies?

For ELISA applications with HRP-conjugated PRDM11 antibodies, researchers should:

• Plate coating: Coat microplates with capture antibody or target protein (if direct ELISA)

• Blocking: Block with appropriate blocking buffer (typically 1-5% BSA)

• Sample incubation: Apply samples containing PRDM11 protein

• Detection: Add HRP-conjugated PRDM11 antibody at optimized dilution

• Substrate reaction: Apply TMB or other appropriate HRP substrate

• Signal measurement: Measure absorbance at appropriate wavelength after stopping reaction

Researchers should perform preliminary titration experiments to determine optimal antibody concentration that provides maximum specific signal with minimal background . For quantitative analysis, standard curves using recombinant PRDM11 protein are essential.

Why might detected PRDM11 molecular weight differ from predicted weight?

The observed molecular weight of PRDM11 in Western blotting (approximately 58 kDa) may differ from the calculated weight (53-57 kDa) due to several factors :

• Post-translational modifications: Glycosylation, phosphorylation, or other modifications can significantly alter protein mobility

• Alternative splicing: Different splice variants may be detected in different tissues

• Protein conformations: Incompletely denatured proteins may migrate differently

• Sample preparation conditions: Reducing vs. non-reducing conditions can affect mobility

• Multiple modified forms: If a protein has different modified forms simultaneously, multiple bands may be detected

When observed weight differs from expected, researchers should confirm identity through additional methods such as mass spectrometry or immunoprecipitation followed by Western blotting.

What are the common sources of background in PRDM11 immunostaining and how can they be minimized?

When performing immunohistochemistry or immunofluorescence with PRDM11 antibodies, background issues may arise from:

• Insufficient blocking: Increase blocking agent concentration (5-10% normal serum) and/or time

• Cross-reactivity: Pre-absorb antibody with proteins from species similar to target

• Endogenous peroxidase activity: For HRP-conjugated antibodies, pre-treat samples with hydrogen peroxide solution

• Endogenous biotin: When using biotin-based detection systems, block endogenous biotin

• Fixation artifacts: Optimize fixation protocols (duration, fixative concentration)

• Antibody concentration: Titrate antibody to determine optimal concentration

• Non-specific binding: Include detergents (0.1-0.3% Triton X-100 or Tween-20) in wash buffers

For HRP-conjugated antibodies specifically, researchers should implement additional peroxidase blocking steps and carefully optimize substrate development times to minimize background while maintaining specific signal .

How should researchers interpret conflicting PRDM11 expression data between different detection methods?

When facing discrepancies between different detection methods (e.g., Western blot vs. immunohistochemistry), researchers should:

• Evaluate epitope accessibility: Different methods expose different epitopes based on protein folding/fixation

• Consider cellular localization: PRDM11 localizes to both cytoplasm and nucleus, so detection method sensitivity to subcellular compartments may vary

• Assess antibody specificity: Confirm that different antibodies target the same region or different regions of PRDM11

• Examine sample preparation differences: Fixation methods for IHC vs. protein extraction for WB affect epitope presentation

• Quantify expression levels: Low expression may be detectable by sensitive methods but not by less sensitive techniques

• Validate with orthogonal approaches: Combine antibody-based methods with mRNA detection or mass spectrometry

Systematic analysis of these factors helps reconcile conflicting data and provides more comprehensive understanding of PRDM11 expression patterns.

How can researchers effectively study PRDM11's potential role in transcriptional regulation?

To investigate PRDM11's transcriptional regulatory functions, researchers should implement:

• Chromatin Immunoprecipitation (ChIP): Use PRDM11 antibodies to identify genomic binding sites

• Reporter gene assays: Test PRDM11's effect on promoter/enhancer activity

• Co-immunoprecipitation: Identify PRDM11 interaction partners in transcriptional complexes

• Methyltransferase activity assays: Assess PRDM11's potential histone methyltransferase function suggested by Gene Ontology annotations

• Gene expression profiling: Compare transcriptomes in PRDM11 overexpression and knockdown models

• Computational analysis: Identify enriched binding motifs in PRDM11-bound genomic regions

This multi-modal approach would parallel successful studies of other PRDM family members, such as PRDM1, which was identified as a master regulator through systematic multiomics analysis .

What are appropriate experimental designs for investigating PRDM11's role in Hermansky-Pudlak Syndrome?

To explore PRDM11's relationship with Hermansky-Pudlak Syndrome 1 (HPS1) , researchers should consider:

• Patient sample analysis: Compare PRDM11 expression and localization in HPS1 patient vs. control samples

• Genetic modulation studies: Assess phenotypic changes in disease models upon PRDM11 knockout/overexpression

• Mechanistic investigations: Examine protein-protein interactions between PRDM11 and known HPS1-associated proteins

• Functional assays: Measure lysosomal function, platelet activity, and melanin synthesis in models with altered PRDM11 expression

• Signaling pathway analysis: Identify pathways affected by PRDM11 modulation in relevant cell types

• Structure-function analysis: Map domains of PRDM11 critical for particular cellular functions

These approaches would establish whether PRDM11's association with HPS1 is causative, consequential, or correlative, providing deeper understanding of disease mechanisms.

What methodological approaches can determine if PRDM11 has biological functions similar to its paralog PRDM7?

To investigate functional relationships between PRDM11 and its paralog PRDM7 , researchers should implement:

• Comparative expression analysis: Map tissue-specific and developmental expression patterns of both proteins

• Domain function analysis: Test whether corresponding domains from each protein have similar biochemical activities

• Interactome comparison: Identify and compare protein interaction partners

• Rescue experiments: Test whether PRDM7 expression can rescue phenotypes in PRDM11-deficient models

• Evolutionary analysis: Examine conservation patterns to identify functionally critical regions

• Double knockout studies: Assess whether simultaneous depletion produces synergistic phenotypes

This systematic approach would clarify whether PRDM7 and PRDM11 have redundant, complementary, or distinct biological functions, informing both basic science and potential therapeutic strategies.

How can researchers effectively use PRDM11 antibodies to study potential epigenetic regulatory functions?

Given PRDM11's potential methyltransferase activity , researchers investigating its epigenetic functions should:

• Histone modification analysis: Use ChIP-seq with histone mark antibodies at PRDM11 binding sites

• In vitro methyltransferase assays: Test PRDM11's activity on histone peptides

• Mass spectrometry: Identify specific residues modified by PRDM11

• Epigenome editing: Recruit PRDM11 to specific genomic loci and assess chromatin changes

• Single-cell approaches: Examine correlation between PRDM11 binding and chromatin states

• Sequential ChIP: Determine co-occupancy of PRDM11 with other chromatin modifiers

These approaches would establish whether PRDM11 directly modifies histones or recruits other epigenetic regulators, similar to the established mechanisms of other PRDM family proteins like PRDM1, which alters chromatin accessibility in T cells .

Data Table: PRDM11 Antibody Properties Comparison