HIST1H1B (Ab-188) Antibody

What is the HIST1H1B (Ab-188) Antibody and what does it target?

The HIST1H1B (Ab-188) Antibody is a rabbit polyclonal antibody that specifically targets human histone H1.5 protein (also known as H1F5). It recognizes a peptide sequence around the serine residue at position 188 of human histone H1.5 . Histone H1.5 is a linker histone that binds to DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. This protein plays critical roles in the condensation of nucleosome chains into higher-order structured fibers and regulates gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation .

How does the HIST1H1B (Ab-188) Antibody differ from the HIST1H1B (Ab-137) Antibody?

These antibodies recognize different epitopes of the same histone H1.5 protein. The HIST1H1B (Ab-188) Antibody targets a peptide sequence around the serine residue at position 188 , while the HIST1H1B (Ab-137) Antibody targets a peptide sequence around the threonine residue at position 137 . This distinction is important for experimental design, as post-translational modifications at or near these sites might affect antibody recognition. For phosphorylation studies, researchers might specifically choose one antibody over the other depending on which site they're investigating, as phosphorylation events can significantly alter histone function in chromatin dynamics .

What applications has HIST1H1B (Ab-188) Antibody been validated for?

The HIST1H1B (Ab-188) Antibody has been validated for several key research applications:

• Western Blotting (WB): Successfully detects a 23 kDa band in various human cell lysates including MCF-7 and U87, with recommended dilutions of 1:100-1:1000 .

• Immunohistochemistry (IHC): Effective for paraffin-embedded human tissue sections, including melanoma samples, at dilutions of 1:20-1:200 .

• Enzyme-Linked Immunosorbent Assay (ELISA): Validated at dilutions of 1:2000-1:10000 .

The antibody has demonstrated specific reactivity with human samples in these applications, making it a reliable tool for investigating histone H1.5 expression and localization in human cells and tissues .

What are the optimal conditions for Western blotting with HIST1H1B (Ab-188) Antibody?

For optimal Western blotting results with HIST1H1B (Ab-188) Antibody:

• Sample preparation: Extract histones using specialized protocols that account for their nuclear localization. Acid extraction methods are often preferred for histone proteins.

• Protein loading: Load 20-30 μg of total protein per lane.

• Gel selection: Use 12-15% polyacrylamide gels to resolve the relatively small histone protein (23 kDa).

• Transfer conditions: Transfer to PVDF membranes at 100V for 1 hour or 30V overnight at 4°C.

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

• Primary antibody: Dilute HIST1H1B antibody to 1.5 μg/ml (approximately 1:500 dilution) in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use anti-rabbit IgG at 1:50000 dilution .

• Detection: The predicted band size for Histone H1.5 is 23 kDa, which aligns with the observed band size in validated cell lines .

The antibody has been successfully used with various human cell lysates including MCF-7 (breast cancer) and U87 (glioblastoma) lines, suggesting broad utility across different cancer and normal cell types .

How should HIST1H1B (Ab-188) Antibody be stored to maintain optimal activity?

For maximum antibody stability and activity:

• Storage temperature: Store the antibody at -20°C for long-term storage.

• Buffer conditions: The antibody is supplied in a preservative solution containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 .

• Aliquoting: Upon receipt, divide the antibody into small single-use aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality.

• Working solution: When preparing a working solution, dilute only the amount needed for immediate use, and store the diluted antibody at 4°C for no more than a week.

• Stability: When properly stored, the antibody should maintain activity for at least one year from the date of receipt.

Maintaining these storage conditions is critical for preserving antibody specificity and sensitivity, especially for chromatin immunoprecipitation (ChIP) and other applications requiring high antibody quality .

What controls should be included when using HIST1H1B (Ab-188) Antibody in experiments?

Proper experimental controls are essential for validating results with HIST1H1B (Ab-188) Antibody:

• Positive control: Include lysates from cell lines known to express histone H1.5, such as U87, MCF-7, or HEK293 cells, which have been validated with this antibody .

• Negative control: Consider cells where H1.5 expression has been knocked down using siRNA/shRNA, though expression is typically ubiquitous in most cell types.

• Loading control: Include detection of a stable reference protein (e.g., GAPDH, β-actin) for Western blotting, or total histone H3 for chromatin studies.

• Isotype control: For IHC or IF experiments, include a rabbit IgG isotype control at the same concentration to assess non-specific binding.

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide (Ser-188 region) should abolish specific signals, confirming antibody specificity.

• Cross-validation: When possible, confirm key findings using the complementary HIST1H1B (Ab-137) Antibody that targets a different epitope, or using alternative methods such as mass spectrometry .

These controls help distinguish true signal from background and validate antibody specificity across different experimental contexts.

How can HIST1H1B (Ab-188) Antibody be used to study histone H1.5's role in chromatin dynamics?

The HIST1H1B antibody is valuable for investigating histone H1.5's role in chromatin structure and dynamics through several advanced approaches:

• Chromatin Immunoprecipitation (ChIP): While not explicitly validated in the documentation, this antibody class can potentially be used for ChIP assays to identify genomic regions where histone H1.5 binds. Typically, a 1:50-1:100 dilution is appropriate for ChIP applications with polyclonal antibodies.

• Fluorescence Recovery After Photobleaching (FRAP): As demonstrated in research with histone H1 proteins, FRAP experiments can reveal the dynamic binding properties of H1.5 to chromatin. Research has shown that the recovery time of H1-GFP after photobleaching is affected by competing chromatin binding proteins like high-mobility group (HMG) proteins .

• Co-immunoprecipitation (Co-IP): This antibody can help identify proteins that interact with histone H1.5, such as DNA methyltransferases or chromatin remodeling complexes.

• Immunofluorescence with super-resolution microscopy: Can reveal the spatial distribution of H1.5 within nuclear domains and its colocalization with other chromatin markers.

Studies have revealed that histone H1 proteins have different mobility rates in euchromatin versus heterochromatin, with stronger binding observed in condensed heterochromatin regions. The t80 (time to reach 80% recovery) for H1-GFP in euchromatin is approximately 100 seconds, while competitive interactions with HMG proteins can reduce this to less than 50 seconds .

What insights can be gained from studying the competition between histone H1.5 and HMG proteins using this antibody?

Research using histone H1 antibodies has revealed important insights about the dynamic interplay between histone H1 and high-mobility group (HMG) proteins in chromatin regulation:

• Competitive binding: All three families of HMG proteins (HMGA, HMGB, and HMGN) weaken the binding of histone H1 to nucleosomes by competing for chromatin binding sites .

• Synergistic effects: The competition between HMG proteins and histone H1 is synergistic rather than merely additive. When multiple HMG family proteins are present, the effect on H1 mobility is greater than the sum of their individual effects .

• Dose-dependent relationship: The ability of HMG proteins to increase histone H1 mobility is dose-dependent, with higher concentrations of HMG proteins causing greater increases in H1 mobility .

• Chromatin context dependence: The competition between HMG proteins and histone H1 varies between euchromatin and heterochromatin domains. The HIST1H1B antibody can be used to analyze these differences through immunofluorescence co-localization studies with euchromatin/heterochromatin markers.

This data from FRAP experiments reveals that injection of HMGB1 protein significantly increased the R20s (recovery at 20 seconds) values for H1°-GFP by approximately 8.2% and decreased the t40 (time to reach 40% recovery) to 7 seconds, demonstrating the competitive dynamics between these chromatin architectural proteins .

Can HIST1H1B (Ab-188) Antibody be used to investigate post-translational modifications of histone H1.5?

While the HIST1H1B (Ab-188) Antibody primarily detects total histone H1.5 protein regardless of modification status, it can still be useful in post-translational modification (PTM) studies:

• Modification-sensitive detection: Since this antibody targets the region around Ser-188, extensive modifications at or near this site might affect antibody binding. This property can be leveraged to indirectly study certain PTMs.

• Sequential immunoprecipitation: The antibody can be used in a first round of immunoprecipitation to capture total H1.5, followed by probing with modification-specific antibodies against phosphorylation, methylation, or acetylation marks.

• Comparative analysis with HIST1H1B (Ab-137): Using both the Ab-188 and Ab-137 antibodies in parallel experiments can provide insights into the modification status of different regions of the protein .

• Mass spectrometry integration: Following immunoprecipitation with HIST1H1B (Ab-188) Antibody, mass spectrometry analysis can identify and quantify various PTMs on the captured histone H1.5 protein.

The Ser-188 site in histone H1.5 is a known target for phosphorylation by cyclin-dependent kinases (CDKs) during the cell cycle. Researchers investigating cell cycle-dependent chromatin changes may find this antibody particularly valuable for studying how H1.5 phosphorylation affects its binding dynamics and function.

What are common issues when using HIST1H1B (Ab-188) Antibody in Western blotting and how can they be resolved?

When working with HIST1H1B (Ab-188) Antibody in Western blotting, researchers may encounter several challenges:

• Multiple bands or non-specific binding:

  • Cause: Cross-reactivity with other histone H1 variants or insufficient blocking

  • Solution: Increase blocking time/concentration, optimize antibody dilution (start with 1:500), or perform additional washing steps

• Weak or no signal:

  • Cause: Insufficient protein extraction or inadequate transfer of histones

  • Solution: Use specialized histone extraction protocols (acid extraction), ensure sufficient loading (30+ μg), and optimize transfer conditions for small proteins (use PVDF membrane and add 0.1% SDS to transfer buffer)

• High background:

  • Cause: Over-development or excessive antibody concentration

  • Solution: Reduce primary antibody concentration to 1:1000, use fresher blocking agent, increase washing time/frequency

• Inconsistent results between experiments:

  • Cause: Histone modifications affecting epitope recognition or variable extraction efficiency

  • Solution: Standardize cell culture conditions, use consistent lysis protocols, and consider the cell cycle phase of samples (histone modifications vary through cell cycle)

When troubleshooting, remember that the expected band size for histone H1.5 is 23 kDa. The antibody has been validated on both MCF-7 and U87 cell lysates at a concentration of 1.5 μg/ml with a secondary antibody dilution of 1:50000 .

How should results from different applications of HIST1H1B (Ab-188) Antibody be integrated for comprehensive analysis?

Integrating data from multiple applications provides a more complete understanding of histone H1.5 biology:

• Correlation of expression and localization data:

  • Western blot data provides quantitative information about total H1.5 expression

  • IHC/IF results reveal spatial distribution within tissues/cells

  • Combined analysis can identify whether changes in total expression correlate with redistribution within the nucleus

• Multi-omics integration:

  • ChIP-seq data reveals genomic binding sites of H1.5

  • RNA-seq can identify genes affected by H1.5 binding

  • Proteomics can identify H1.5 binding partners

  • Cross-referencing these datasets provides insights into H1.5's regulatory mechanisms

• Temporal dynamics analysis:

  • FRAP experiments measure H1.5 binding dynamics in live cells

  • Western blots at different time points track expression changes

  • Combining temporal data provides insights into how H1.5 function changes during processes like differentiation or cell cycle progression

• Comparative analysis with other histone variants:

  • Parallel experiments with antibodies against other H1 variants help distinguish unique roles of H1.5

  • Signal ratios between different variants can be more informative than absolute values

For example, research has shown that histone H1 mobility varies between euchromatin and heterochromatin regions, with recovery times differing significantly. In euchromatin, H1-GFP shows a t80 (time to 80% recovery) of approximately 100 seconds, while in heterochromatin regions, the binding is stronger with longer recovery times .

How can researchers distinguish between specific and non-specific signals when using HIST1H1B (Ab-188) Antibody in IHC?

To distinguish between specific and non-specific signals in immunohistochemistry with HIST1H1B (Ab-188) Antibody:

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (region around Ser-188)

  • Compare with non-competed antibody on sequential tissue sections

  • Signals that disappear in the competed sample represent specific binding

• Cellular localization assessment:

  • Histone H1.5 should show predominant nuclear localization

  • Cytoplasmic staining typically indicates non-specific binding

  • Compare with DAPI nuclear counterstain to confirm nuclear localization

• Dilution series optimization:

  • Test multiple antibody dilutions (from 1:20 to 1:200 as recommended)

  • Specific signals should decrease proportionally with dilution

  • Non-specific background may persist even at high dilutions

• Isotype control:

  • Use rabbit IgG at the same concentration as primary antibody

  • Any signal present in isotype control represents non-specific binding

• Tissue-specific considerations:

  • Include positive control tissues known to express H1.5 (most nucleated cells)

  • For human melanoma samples, a 1:100 dilution has been validated as optimal

  • Interpret staining patterns in the context of known biology (e.g., stronger staining in actively dividing cells)

• Signal quantification:

  • Use digital image analysis to quantify nuclear staining intensity

  • Compare signal-to-background ratios across different conditions

  • Statistical analysis of multiple fields/samples strengthens confidence in results

How can HIST1H1B (Ab-188) Antibody be used to investigate the role of histone H1.5 in cancer biology?

The HIST1H1B (Ab-188) Antibody offers several approaches to investigate histone H1.5's role in cancer biology:

• Expression profiling across cancer types:

  • Compare H1.5 levels between tumor and matched normal tissues using Western blotting and IHC

  • The antibody has been validated in cancer cell lines including MCF-7 (breast cancer) and U87 (glioblastoma), as well as in human melanoma tissue sections

  • Quantitative analysis can establish correlations between H1.5 expression and clinical outcomes

• Chromatin structure analysis in tumor cells:

  • Use IF/IHC to examine changes in nuclear distribution of H1.5 during tumor progression

  • Analyze colocalization with heterochromatin/euchromatin markers to assess global chromatin changes

  • Studies have shown different H1 mobility in euchromatin versus heterochromatin regions, which may be altered in cancer cells

• Functional role in oncogene regulation:

  • Combine ChIP (using this antibody) with gene expression analysis to identify cancer-relevant genes regulated by H1.5

  • Investigate how H1.5 distribution changes in response to oncogenic signaling pathways

• Interaction with cancer-specific HMG proteins:

  • Cancer cells often overexpress HMG proteins, which compete with H1 for chromatin binding

  • Research has shown synergistic effects when multiple HMG families are present, potentially enhancing chromatin accessibility in cancer cells

  • The antibody can be used in co-immunoprecipitation studies to identify cancer-specific interaction partners

The antibody's application in melanoma tissue sections at 1:100 dilution has already demonstrated its utility in cancer research contexts , providing a foundation for broader applications across different tumor types.

What methodological considerations are important when using HIST1H1B (Ab-188) Antibody in chromatin immunoprecipitation (ChIP) experiments?

While not explicitly validated for ChIP in the documentation, polyclonal antibodies against histone proteins are commonly used in this application with specific considerations:

• Crosslinking optimization:

  • Histone H1 has both nucleosome-bound and free-floating populations

  • Standard 1% formaldehyde crosslinking (10 minutes at room temperature) is typically sufficient

  • Dual crosslinking with both formaldehyde and protein-specific crosslinkers like DSG (disuccinimidyl glutarate) can improve histone H1 ChIP efficiency

• Sonication parameters:

  • Chromatin should be sheared to 200-500 bp fragments

  • Over-sonication can disrupt histone-DNA interactions

  • Verify sonication efficiency by agarose gel electrophoresis before proceeding

• Antibody amount optimization:

  • Start with 5 μg antibody per ChIP reaction

  • Perform titration experiments to determine optimal antibody:chromatin ratio

  • Include IgG control at the same concentration

• Blocking strategy:

  • Pre-clear chromatin with protein A/G beads and rabbit IgG

  • Use BSA and salmon sperm DNA in blocking buffer to reduce background

• Washing stringency balance:

  • Histone H1 has both specific and non-specific DNA interactions

  • Too stringent washing may remove legitimate interactions

  • Start with standard wash buffers and adjust salt concentration based on results

• Validation with known targets:

  • Include ChIP-qPCR for regions known to be enriched or depleted for H1.5

  • Benchmark against published H1.5 ChIP-seq datasets

  • Consider parallel ChIP with both Ab-188 and Ab-137 antibodies to validate findings

• Data normalization considerations:

  • Normalize to input rather than IgG control

  • Consider using spike-in controls for quantitative comparisons between conditions

How does the dynamic competition between histone H1.5 and HMG proteins affect experimental design and data interpretation?

The dynamic competition between histone H1.5 and HMG proteins has significant implications for experimental design and interpretation:

• Experimental timing considerations:

  • HMG protein expression fluctuates during cell cycle and differentiation

  • The competition effect is dose-dependent, with higher HMG concentrations causing greater H1 mobility

  • Synchronize cells or document cell cycle phase when comparing H1.5 binding across conditions

• Contextual interpretation of results:

  • H1.5 binding patterns should be interpreted in the context of HMG protein levels

  • The R20s (recovery at 20 seconds) of H1-GFP increases by approximately 8-12% upon introduction of HMG proteins

  • Parallel quantification of major HMG proteins provides context for H1.5 binding changes

• Synergistic effects in multi-protein systems:

  • The HMG protein families affect H1 binding synergistically rather than competitively among themselves

  • When HMGB1 and HMGN2 are combined, the increase in H1 mobility (8.7%) is greater than the sum of their individual effects (approximately 4%)

  • This synergy should be considered when manipulating expression of single HMG proteins

• Chromatin domain-specific analysis:

  • Competition effects differ between euchromatin and heterochromatin

  • In euchromatin, HMG proteins can decrease H1 recovery time (t80) from 100s to less than 50s

  • Domain-specific analysis (rather than whole-nucleus) provides more accurate insights

• Methodological implications:

  • FRAP and other live-cell imaging techniques are essential to capture the dynamic nature of these interactions

  • Fixed-cell techniques like ChIP or IF provide only snapshots of a highly dynamic system

  • Combining multiple methodologies is crucial for comprehensive understanding

This data highlights how HMG proteins significantly reduce H1 binding time (lower t40) and increase recovery percentage (higher R20s), demonstrating their role in modulating chromatin accessibility .