Phospho-HIST1H1E (T3) Antibody

What is HIST1H1E and what is the significance of T3 phosphorylation?

HIST1H1E, also known as Histone H1.4, is a linker histone that helps maintain higher-order chromatin structure by binding to nucleosome entry and exit sites. This protein belongs to the H1 histone family and is encoded by the HIST1H1E gene (Gene ID: P10412) . Phosphorylation at threonine 3 (T3) is a critical post-translational modification that regulates chromatin compaction and DNA accessibility during various cellular processes.

The phosphorylation of T3 in HIST1H1E is regulated dynamically during the cell cycle, similar to the well-characterized H3T3 phosphorylation. While H3T3 phosphorylation is known to be regulated by the Haspin kinase and dephosphorylated by the PP1γ/Repo-Man complex , the exact kinases and phosphatases regulating HIST1H1E T3 phosphorylation are still being investigated. This modification likely plays roles in chromosome condensation, transcriptional regulation, and DNA damage response pathways.

How should Phospho-HIST1H1E (T3) antibodies be stored and handled?

Based on manufacturer specifications, optimal storage and handling procedures for Phospho-HIST1H1E (T3) antibodies include:

Researchers should avoid repeated freeze-thaw cycles as this can compromise antibody activity. Upon receipt, it is recommended to divide the antibody into small aliquots before freezing. When handling the antibody during experiments, keep it on ice and return to the appropriate storage temperature promptly after use.

What experimental applications is Phospho-HIST1H1E (T3) antibody suitable for?

The Phospho-HIST1H1E (T3) antibody has been validated for multiple applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): Can detect phosphorylated HIST1H1E in protein lysates .

• Immunofluorescence (IF): Suitable for visualizing the subcellular localization of phosphorylated HIST1H1E in fixed cells .

• Western Blotting (WB): While not explicitly mentioned for the T3 phospho-antibody, similar phospho-specific histone antibodies are commonly used for WB applications to detect phosphorylation levels.

• Chromatin Immunoprecipitation (ChIP): Though not specifically validated, phospho-histone antibodies are frequently employed in ChIP experiments to map genomic locations.

For optimal results, researchers should follow specific protocols recommended by the antibody manufacturer for each application, including appropriate dilutions, incubation times, and detection methods.

How can researchers optimize immunostaining protocols using Phospho-HIST1H1E (T3) antibody?

Optimizing immunostaining protocols with Phospho-HIST1H1E (T3) antibody requires careful consideration of several technical parameters:

Fixation Methods:

• Paraformaldehyde (4%) fixation for 10-15 minutes is typically suitable for preserving phospho-epitopes while maintaining cell morphology.

• Methanol fixation may result in epitope masking and is generally not recommended for phospho-specific antibodies.

• Include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) in all buffers to prevent dephosphorylation during sample processing.

Blocking and Permeabilization:

• Use 5% BSA in PBS with 0.1-0.3% Triton X-100 for effective blocking and permeabilization.

• Consider adding 5-10% normal serum from the species of the secondary antibody to reduce background.

Antibody Incubation:

• Primary antibody dilutions should be empirically determined, starting with manufacturer recommendations.

• Incubate at 4°C overnight for optimal signal-to-noise ratio.

• Include phosphatase inhibitors in antibody dilution buffers.

Signal Amplification Strategies:

• For low abundance phospho-epitopes, consider using biotin-streptavidin amplification systems.

• Tyramide signal amplification (TSA) can enhance detection of weak signals.

Controls:

• Include phosphatase-treated samples as negative controls to validate phospho-specificity.

• Use peptide competition assays to confirm signal specificity.

What approaches can address cross-reactivity concerns with other phosphorylated histones?

Addressing cross-reactivity is crucial for generating reliable data with phospho-specific antibodies:

• Peptide Competition Assays: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides. Signal reduction with phosphorylated peptide but not with non-phosphorylated peptide confirms specificity.

• Phosphatase Treatment Controls: Compare staining patterns between untreated samples and those treated with lambda phosphatase or similar. Significant signal reduction indicates phospho-specificity.

• Knockout/Knockdown Validation: Use HIST1H1E knockdown cells or tissues to confirm signal specificity.

• Cross-Validation Methods: Compare results with alternative detection methods:

  • Mass spectrometry to confirm phosphorylation status

  • Alternative antibodies targeting the same modification

  • Phospho-specific staining methods

• Antibody Characterization Table:

How can Phospho-HIST1H1E (T3) antibody be used to study chromatin dynamics during cell cycle?

Studying chromatin dynamics using Phospho-HIST1H1E (T3) antibody requires careful experimental design:

Cell Synchronization Protocols:

• Double Thymidine Block: For G1/S boundary synchronization

• Nocodazole treatment: For mitotic arrest

• Serum starvation-release: For G0/G1 transition

Experimental Approach:

• Synchronize cells at different cell cycle stages

• Fix cells and immunostain with Phospho-HIST1H1E (T3) antibody

• Co-stain with cell cycle markers (e.g., pH3S10 for mitosis, PCNA for S phase)

• Quantify phosphorylation signal intensity relative to total HIST1H1E

• Correlate phosphorylation patterns with chromatin conformational changes

Data Analysis Methods:

• High-content imaging for single-cell analysis

• Flow cytometry for population-level quantification

• Time-lapse microscopy for dynamic phosphorylation tracking

The PP1γ/Repo-Man complex has been identified as a regulator of histone H3 phosphorylation at T3 . Researchers can investigate whether similar phosphatase complexes regulate HIST1H1E T3 phosphorylation by employing phosphatase inhibitors and monitoring changes in phosphorylation patterns throughout the cell cycle.

What techniques can be used to study the relationship between T3 phosphorylation and other HIST1H1E modifications?

Studying the interplay between T3 phosphorylation and other post-translational modifications (PTMs) on HIST1H1E requires sophisticated methodological approaches:

Experimental Strategies:

• Sequential Chromatin Immunoprecipitation (Re-ChIP):

  • First ChIP with Phospho-HIST1H1E (T3) antibody

  • Second ChIP with antibodies against other modifications

  • Analyze enriched genomic regions to identify co-occurrence

• Mass Spectrometry-Based Approaches:

  • Immunoprecipitate HIST1H1E and analyze by tandem mass spectrometry

  • Quantify co-occurrence of multiple modifications on the same HIST1H1E molecule

  • Use SILAC or TMT labeling for quantitative comparisons across conditions

• Proximity Ligation Assay (PLA):

  • Detect spatial proximity (<40 nm) between T3 phosphorylation and other modifications

  • Provides single-cell resolution of modification co-occurrence

• Multicolor Immunofluorescence:

  • Use antibodies against different modifications with spectrally distinct fluorophores

  • Perform colocalization analysis to identify regions enriched for multiple modifications

The C-terminal tail of HIST1H1E has been implicated in cellular senescence and aging , suggesting potential functional interactions between C-terminal modifications and T3 phosphorylation. Researchers could investigate whether T3 phosphorylation status affects the function of the C-terminal domain in chromatin compaction.

How can researchers troubleshoot common issues with Phospho-HIST1H1E (T3) antibody?

Detailed Troubleshooting Approaches:

• For Weak Signals:

  • Increase antibody concentration incrementally

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try alternative antigen retrieval methods

  • Use signal amplification systems (TSA, polymer-based detection)

  • Ensure phosphatase inhibitors are present in all buffers

• For High Background:

  • Increase blocking time and concentration (5-10% BSA or normal serum)

  • Add 0.1-0.3% Triton X-100 to washing buffers

  • Pre-absorb antibody with cell/tissue lysates from non-target species

  • Reduce secondary antibody concentration

  • Include extra washing steps with higher salt concentration

• For Inconsistent Results:

  • Standardize cell culture conditions and treatments

  • Create detailed protocols with precise timing for each step

  • Prepare fresh buffers for each experiment

  • Maintain consistent imaging parameters

What are the optimal sample preparation methods for different experimental applications?

Western Blotting:

• Harvest cells in ice-cold PBS containing phosphatase inhibitors

• Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors

• Sonicate briefly to shear chromatin and release histone proteins

• Centrifuge at high speed to clear debris

• Determine protein concentration by Bradford or BCA assay

• Denature proteins at 95°C for 5 minutes in Laemmli buffer

• Load 10-30 μg protein per lane on SDS-PAGE gel

Immunofluorescence:

• Grow cells on glass coverslips or chamber slides

• Wash with PBS containing phosphatase inhibitors

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

• Permeabilize with 0.3% Triton X-100 for 10 minutes

• Block with 5% BSA in PBS for 1 hour

• Incubate with primary antibody overnight at 4°C

• Apply appropriate secondary antibody for 1-2 hours at room temperature

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

ELISA:

• Coat microplate wells with target protein or peptide

• Block non-specific binding sites

• Add diluted Phospho-HIST1H1E (T3) antibody

• Wash and add HRP-conjugated secondary antibody

• Develop with TMB substrate and measure absorbance

How can Phospho-HIST1H1E (T3) antibody contribute to aging and senescence research?

The study of histone modifications in aging and cellular senescence represents an important research area. The C-terminal tail of HIST1H1E has been linked to cellular senescence and premature aging , suggesting that phosphorylation at T3 might also play a role in these processes.

Experimental Approaches:

• Comparative Analysis:

  • Compare T3 phosphorylation patterns between young and senescent cells

  • Analyze T3 phosphorylation in tissues from young versus aged organisms

  • Study T3 phosphorylation in premature aging disorders

• Functional Studies:

  • Investigate how T3 phosphorylation affects HIST1H1E binding to chromatin

  • Examine changes in gene expression patterns correlated with T3 phosphorylation status

  • Create phosphomimetic (T3E) and phospho-dead (T3A) HIST1H1E mutants to study functional consequences

• Therapeutic Implications:

  • Screen for compounds that modulate T3 phosphorylation

  • Investigate whether existing senolytic drugs affect HIST1H1E phosphorylation

Research has shown that cells expressing mutant HIST1H1E proteins have dramatically reduced proliferation rates and undergo accelerated senescence . Investigating the role of T3 phosphorylation in this context could provide insights into the molecular mechanisms of aging and potential therapeutic targets.

What is the relationship between HIST1H1E T3 phosphorylation and phosphatase activity?

Understanding the regulation of HIST1H1E T3 phosphorylation requires investigation of relevant phosphatases. Research on histone H3 has identified PP1γ/Repo-Man as a phosphatase complex that targets H3T3ph , suggesting similar mechanisms might regulate HIST1H1E.

Experimental Approaches:

• Phosphatase Inhibitor Studies:

  • Treat cells with okadaic acid (PP1/PP2A inhibitor) or other phosphatase inhibitors

  • Monitor changes in HIST1H1E T3 phosphorylation levels

  • Correlate inhibitor specificity with phosphorylation patterns

• Candidate Phosphatase Screening:

  • Perform siRNA knockdown of candidate phosphatases

  • Overexpress phosphatases and assess effects on T3 phosphorylation

  • Conduct in vitro dephosphorylation assays using purified phosphatases

• Phosphohistidine Phosphatase Analysis:

  • Apply screening methods for phosphohistidine phosphatase activity

  • Test whether PHPT1 or related phosphatases can dephosphorylate HIST1H1E at T3

  • Compare with known substrates like histone H4

A methodology similar to that used for studying phosphohistidine phosphatase activity could be adapted for investigating enzymes that regulate HIST1H1E T3 phosphorylation . This includes chemical phosphorylation of substrate peptides, removal of excess phosphoramidate by anion exchange chromatography, and subsequent dephosphorylation assays.