PHF21A Antibody, HRP conjugated

What is PHF21A and what cellular functions does it perform?

PHF21A, also known as BRAF35-HDAC complex protein BHC80, is a 680 amino acid nuclear protein containing one PHD-type zinc finger and one A.T hook DNA-binding domain. It functions as a key component of the BHC complex, which represses transcription of neuron-specific genes in non-neuronal cells . PHF21A specifically binds to unmethylated histone H3 lysine 4 (H3K4me0) and participates in the lysine-specific demethylase 1 (LSD1) demethylase complex, implicating it as a critical regulatory protein in histone-methylation dynamics . The protein acts as a scaffold within the BHC complex, which functions as a chromatin modifier that deacetylates and demethylates specific sites on histones . PHF21A is predominantly expressed in brain tissue, with three isoforms existing due to alternative splicing events .

What are the advantages of using HRP-conjugated PHF21A antibodies over unconjugated versions?

HRP-conjugated PHF21A antibodies offer several methodological advantages in research applications:

• Direct detection capability without requiring secondary antibodies, simplifying experimental workflows

• Enhanced sensitivity in Western blotting, ELISA, and immunohistochemistry applications

• Reduction in background signals that can occur with two-antibody detection systems

• More consistent results due to standardized enzyme-to-antibody ratios

• Time savings in experimental protocols by eliminating secondary antibody incubation steps

When selecting between conjugated and unconjugated antibodies, researchers should consider that while unconjugated antibodies like those described in the literature (12127-1-AP, E-AB-52472) offer flexibility with different detection systems , HRP-conjugated versions provide workflow efficiency advantages for specific applications where direct enzymatic detection is beneficial.

What are the typical applications for PHF21A HRP-conjugated antibodies?

PHF21A antibodies, including HRP-conjugated versions, are suitable for multiple applications in epigenetics and neuroscience research:

HRP-conjugated PHF21A antibodies are particularly valuable when studying its role in the LSD1 demethylase complex, its interaction with unmethylated H3K4, and its function in transcriptional regulation of neuronal genes .

What experimental controls should be included when using PHF21A HRP-conjugated antibodies?

A robust experimental design incorporating proper controls is essential when using PHF21A HRP-conjugated antibodies:

• Positive Control: Use samples known to express PHF21A, such as HEK-293 cells which have been verified to express detectable levels of the protein .

• Negative Control: Include samples where PHF21A is absent or knockdown/knockout samples if available. For immunohistochemistry, omit primary antibody to assess non-specific binding of detection reagents.

• Blocking Peptide Control: When available, pre-incubation of the antibody with its immunizing peptide should abolish specific signals.

• Cross-Reactivity Control: Test samples from species not expected to react with the antibody based on sequence homology.

• Loading Control: For Western blotting, include antibodies against housekeeping proteins (β-actin, GAPDH) to normalize expression levels.

These controls help validate antibody specificity and ensure experimental rigor when studying PHF21A's role in the BHC complex and its function in transcriptional repression .

How should sample preparation be optimized for detecting PHF21A with HRP-conjugated antibodies?

Optimal sample preparation is crucial for successful detection of PHF21A using HRP-conjugated antibodies:

For Western Blotting:

• Use cell lysis buffers containing protease inhibitors to prevent degradation of PHF21A

• Include phosphatase inhibitors if studying post-translational modifications

• Denature samples at 95°C for 5 minutes in Laemmli buffer containing SDS and DTT

• Load adequate protein (20-50 μg of total protein) to detect the 75 kDa PHF21A band

• Transfer proteins to PVDF membranes (preferred over nitrocellulose for nuclear proteins)

For Immunohistochemistry:

• Formalin-fixed paraffin-embedded sections should be subjected to heat-induced epitope retrieval

• Optimal dilution range of 1:40-1:200 has been validated for PHF21A antibodies

• For brain tissue, where PHF21A is predominantly expressed, extend blocking steps to reduce background

For Chromatin Immunoprecipitation:
Since PHF21A binds to unmethylated H3K4 and functions in the LSD1 complex , mild crosslinking conditions are recommended to preserve protein-DNA interactions while allowing antibody access to epitopes.

What are the recommended storage and handling conditions for PHF21A HRP-conjugated antibodies?

Proper storage and handling of PHF21A HRP-conjugated antibodies is essential to maintain their activity:

• Storage Temperature: Store at -20°C. PHF21A antibodies are typically stable for one year after shipment when properly stored .

• Buffer Composition: PHF21A antibodies are often supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 or phosphate buffered solution containing 0.05% stabilizer and 50% glycerol .

• Aliquoting: For antibodies stored at -20°C, aliquoting may be unnecessary , but is recommended for HRP-conjugated versions to avoid freeze-thaw cycles that can reduce enzymatic activity.

• Freeze-Thaw Cycles: Avoid repeated freeze-thaw cycles which can degrade both the antibody and the HRP conjugate .

• Handling Upon Receipt: When shipped with ice packs, store immediately at the recommended temperature upon receipt .

• Working Dilutions: Prepare fresh working dilutions on the day of the experiment to ensure optimal HRP activity.

Why might I observe high background when using PHF21A HRP-conjugated antibodies, and how can I minimize it?

High background is a common issue when using HRP-conjugated antibodies including those targeting PHF21A. Several strategies can address this problem:

Causes and Solutions for High Background:

• Insufficient Blocking:

  • Increase blocking time from 1 to 2 hours

  • Try alternative blocking agents (5% BSA, 5% non-fat dry milk, commercial blockers)

  • For brain tissue, where PHF21A is predominantly expressed , optimize blocking carefully

• Antibody Concentration Too High:

  • Perform a dilution series starting from 1:500 to 1:2000 to determine optimal concentration

  • For IHC applications, start with the validated range of 1:40-1:200

• Non-specific HRP Activity:

  • Include 0.05% hydrogen peroxide in methanol pretreatment for IHC

  • Add 0.01% sodium azide to blocking buffer (note: not compatible with HRP detection)

• Cross-reactivity:

  • Pre-absorb antibody with non-specific proteins

  • For WB, increase wash duration and number of washes

• Sample-Specific Issues:

  • For tissues with high endogenous peroxidase activity, include a peroxidase quenching step

  • Adjust fixation time for IHC samples

Each experiment may require specific optimization based on the nature of samples and the specificity of the PHF21A antibody used.

How can I troubleshoot weak or absent signals when using PHF21A HRP-conjugated antibodies?

When facing weak or absent signals with PHF21A HRP-conjugated antibodies, consider the following troubleshooting approaches:

Weak/No Signal Troubleshooting Guide:

For Western blot applications specifically, verified protocols using PHF21A antibodies have demonstrated successful detection in HEK-293 cells , making these useful positive controls for troubleshooting.

How can the specificity of PHF21A HRP-conjugated antibodies be validated?

Validating antibody specificity is crucial for obtaining reliable research results. For PHF21A HRP-conjugated antibodies, consider these validation approaches:

• Molecular Weight Verification: Confirm detection at the expected molecular weight of 75 kDa, which corresponds to the calculated molecular weight of PHF21A (680 amino acids) .

• Gene Silencing: Compare signals between wild-type samples and those where PHF21A has been knocked down using siRNA or CRISPR-Cas9 approaches.

• Recombinant Protein Expression: Overexpress tagged PHF21A in cell lines and confirm detection at increased levels and at the correct molecular weight.

• Mass Spectrometry Correlation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the captured protein.

• Multiple Antibody Comparison: Compare results using different antibodies targeting distinct epitopes of PHF21A (such as N-terminal versus C-terminal regions) .

• Blocking Peptide Experiment: Pre-incubate antibody with immunizing peptide (such as the KLH-conjugated synthetic peptide derived from human PHF21A ) to demonstrate signal abrogation.

• Tissue Expression Pattern: Confirm higher expression in brain tissue, where PHF21A is predominantly expressed .

These validation methods ensure that experimental findings accurately reflect PHF21A biology and function.

How can PHF21A antibodies be utilized to study its role in neurodevelopmental disorders?

PHF21A has been implicated in neurodevelopmental disorders, making antibodies against this protein valuable research tools:

PHF21A disruption is associated with intellectual developmental disorder with behavioral abnormalities and craniofacial dysmorphism, with or without seizures . As of 2024, at least 32 people with PHF21A-related syndrome have been identified in medical clinics since the first case was described in 2012 .

Research Applications for PHF21A Antibodies in Neurodevelopmental Studies:

• Expression Analysis in Patient-Derived Samples:

  • PHF21A antibodies can be used to compare protein levels in patient-derived cells vs. controls

  • IHC applications (dilution 1:40-1:200) can assess expression patterns in brain tissue

• Functional Studies of PHF21A Haploinsufficiency:

  • In lymphoblastoid cell lines from translocation subjects with disrupted PHF21A, researchers observed derepression of the neuronal gene SCN3A and reduced LSD1 occupancy at the SCN3A promoter

  • HRP-conjugated antibodies can facilitate detection in functional assays examining similar mechanisms

• Investigation of PHF21A Splicing Variants:

  • Neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development

  • Different antibodies may recognize specific isoforms based on their epitope regions (e.g., AA 351-450)

• Chromatin Modification Studies:

  • PHF21A is part of the BHC complex that acts as a chromatin modifier, deacetylating and demethylating specific histone sites

  • ChIP assays using PHF21A antibodies can map its genomic binding sites in normal vs. pathological conditions

• Protein Complex Analysis:

  • Co-immunoprecipitation with PHF21A antibodies can identify interaction partners in the BHC complex

  • Changes in these interactions may contribute to neurodevelopmental phenotypes

What methodological considerations apply when using PHF21A antibodies in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation (ChIP) with PHF21A antibodies requires specific considerations due to PHF21A's role as a chromatin modifier:

• Crosslinking Optimization:

  • Since PHF21A specifically binds unmethylated histone H3K4 (H3K4me0) , use mild formaldehyde crosslinking (0.5-1%) to preserve protein-DNA interactions

  • Over-crosslinking can mask epitopes in the PHD finger domain that is critical for histone binding

• Sonication Parameters:

  • Optimize sonication to generate 200-500 bp fragments

  • Excessive sonication may disrupt PHF21A-containing protein complexes

• Antibody Selection:

  • Choose antibodies targeting regions not involved in DNA or chromatin binding

  • Verify that the antibody epitope (e.g., AA 351-450) does not overlap with the PHD-type zinc finger or A.T hook DNA-binding domains

• Controls:

  • Include input controls, IgG controls, and positive controls (known PHF21A binding sites)

  • For studies of the LSD1-PHF21A complex, consider parallel ChIP for LSD1 to confirm co-occupancy

• Sequential ChIP (Re-ChIP):

  • Consider sequential ChIP with antibodies against other BHC complex components

  • This approach can identify genomic locations where PHF21A functions within the complete complex

• Analysis of Neuronal vs. Non-neuronal Cells:

  • PHF21A represses transcription of neuron-specific genes in non-neuronal cells

  • Compare binding profiles between these cell types to identify differentially regulated targets

• Data Integration:

  • Correlate ChIP-seq data with RNA-seq to connect PHF21A binding with transcriptional outcomes

  • This approach has revealed that PHF21A disruption leads to derepression of neuronal genes like SCN3A

How do neuronal and non-neuronal PHF21A isoforms differ, and how can antibodies help distinguish them?

Recent research has revealed important differences between neuronal and non-neuronal PHF21A isoforms that can be studied using specific antibodies:

PHF21A undergoes neuron-specific microexon splicing, with the neuronal microexon interfering with nucleosome binding . The temporal expression patterns of these isoforms play crucial roles during brain development:

• Isoform Expression Patterns:

  • Neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development

  • This asynchronous splicing results in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation

• Antibody Selection for Isoform Discrimination:

  • Antibodies targeting regions spanning or adjacent to the neuronal microexon can help distinguish isoforms

  • Epitope mapping is crucial - antibodies targeting AA 351-450 may recognize both isoforms depending on microexon location

• Methodological Approaches:

  • Western blotting with high-resolution gels can separate isoforms with small molecular weight differences

  • Isoform-specific antibodies can be used in IHC to map expression patterns in developing brain tissue

• Functional Studies:

  • Since the PHF21A neuronal microexon interferes with nucleosome binding , different antibodies may show altered immunoprecipitation efficiency depending on chromatin interaction status

  • Co-immunoprecipitation experiments can reveal isoform-specific protein interactions

• Developmental Timing Analysis:

  • Antibodies can track the temporal expression switch from non-neuronal to neuronal isoforms during development

  • This transition represents a critical regulatory mechanism in neurodevelopment

Understanding these isoform differences is crucial for studies of PHF21A's role in neurodevelopmental disorders and for developing targeted therapeutic approaches.