HIST1H1C (Ab-118) Antibody

What is the biological function of HIST1H1C protein?

HIST1H1C (H1.2) is one of the linker histone H1 proteins that bind to nucleosomes and facilitate chromatin compaction. In humans, it is encoded by one of ten different genes expressing linker histones. HIST1H1C functions as a transcriptional repressor by limiting chromatin accessibility, contributing to genome organization and gene expression regulation. Recent research has revealed HIST1H1C acts as a tumor suppressor, whose mutation can drive malignant transformation primarily through three-dimensional genome reorganization, followed by epigenetic reprogramming and derepression of developmentally silenced genes .

How does HIST1H1C expression vary across different cell types?

HIST1H1C expression demonstrates tissue-specific and developmental regulation. In particular, germinal center B-cells (GCB-cells) exhibit 2-4 fold higher expression of HIST1H1C compared to naïve B-cells . This differential expression suggests specialized roles in various cellular contexts. Researchers investigating HIST1H1C should consider these expression patterns when designing experiments and interpreting results across different cellular models.

What epitope does the HIST1H1C (Ab-118) antibody recognize?

The HIST1H1C (Ab-118) antibody is a rabbit polyclonal antibody that recognizes a peptide sequence around site of Lysine (118) derived from Human Histone H1.2 . This specificity is important for researchers to consider when designing experiments, as it determines what form or modification state of the protein will be detected.

What are the validated applications for the HIST1H1C (Ab-118) antibody?

The HIST1H1C (Ab-118) antibody has been validated for several applications:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunofluorescence (IF) at dilutions of 1:50-200

• Immunohistochemistry-Paraffin (IHC-P) at dilutions of 1:20-200

This antibody reacts specifically with human HIST1H1C and has been successfully used on paraffin-embedded human colon cancer tissue and Hela cells as demonstrated in the product validation images .

How should samples be prepared for immunofluorescence studies using HIST1H1C (Ab-118) antibody?

For optimal immunofluorescence results with the HIST1H1C (Ab-118) antibody, researchers should follow these methodological steps:

• Transfer cells to gelatin-coated microscope slides by cytospin (300xg, 10 min)

• Fix with 4% formaldehyde solution for 20 minutes at 21°C

• Wash with PBS

• Block with 10% blocking serum supplemented with 3% Triton X-100 for 45 minutes at 21°C

• Wash and incubate with primary antibody at a dilution of 1:50-200

• Dilute antibody in PBS supplemented with 1.5% blocking serum, 0.3% Triton X-100, and 0.01% sodium azide

• Incubate overnight at 4°C

• Wash and add appropriate secondary fluorescent antibodies for 1 hour at room temperature

Including appropriate controls is essential; use isotype-matched control antibodies as negative controls to confirm specificity of staining .

Can the HIST1H1C (Ab-118) antibody be used in ChIP experiments to study chromatin-associated functions?

While the HIST1H1C (Ab-118) antibody is not specifically validated for ChIP in the provided information, related HIST1H1C antibodies have been used successfully in chromatin immunoprecipitation experiments . For researchers interested in using this antibody for ChIP:

• Cross-link chromatin with 1% formaldehyde

• Sonicate to generate DNA fragments of 200-1000bp

• Immunoprecipitate using the HIST1H1C antibody optimized at concentrations determined through titration experiments

• Include appropriate controls (IgG negative control and a positive control antibody)

• Analyze enrichment through qPCR or next-generation sequencing

Researchers should first conduct validation experiments to determine optimal antibody concentration and conditions for their specific ChIP protocol before proceeding with full experiments.

What are common sources of background when using HIST1H1C (Ab-118) antibody in immunostaining?

High background in immunostaining with HIST1H1C (Ab-118) antibody can result from several factors:

• Insufficient blocking: Increase blocking time or use a higher concentration of blocking serum

• Excessive primary antibody concentration: Optimize by testing a dilution series (1:20-1:200)

• Cross-reactivity with other histone proteins: Include specificity controls

• Inadequate washing: Increase wash duration and number of washes

• Fixation artifacts: Optimize fixation time and conditions

To minimize background, researchers should:

• Ensure complete blocking with 10% blocking serum supplemented with Triton X-100

• Carefully titrate antibody concentration

• Include appropriate negative controls such as isotype-matched control antibodies

• Perform extensive washing steps between antibody incubations

How should the HIST1H1C (Ab-118) antibody be stored to maintain its activity?

For optimal performance, the HIST1H1C (Ab-118) antibody should be:

• Maintained refrigerated at 2-8°C for up to 2 weeks for regular use

• Stored at -20°C in small aliquots for long-term storage to prevent freeze-thaw cycles

• Preserved in buffer containing 0.03% Proclin 300 and 50% Glycerol

Multiple freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency. It's recommended to make small aliquots when first receiving the antibody to minimize this issue.

How can HIST1H1C (Ab-118) antibody be used to investigate the role of HIST1H1C in lymphoma development?

Given that HIST1H1C mutations are highly recurrent in B-cell lymphomas and act as genetic driver mutations, the HIST1H1C (Ab-118) antibody can be utilized to:

• Analyze HIST1H1C protein levels and localization in lymphoma patient samples through IHC-P

• Compare HIST1H1C expression between normal B-cells and lymphoma cells

• Investigate changes in HIST1H1C distribution in chromatin following mutation

• Study co-localization with other factors involved in chromatin modification

• Examine correlations between HIST1H1C expression/localization and patient outcomes

Researchers should consider:

• Using tissue microarrays containing multiple lymphoma samples and normal controls

• Combining HIST1H1C staining with markers of chromatin state (H3K27me3, H3K36me2)

• Correlating protein data with genomic mutation analysis of HIST1H1C

How does HIST1H1C loss affect chromatin structure and gene expression in lymphoma?

Loss of HIST1H1C function in lymphoma results in:

• Profound architectural remodeling of the genome characterized by large-scale shifts of chromatin from compacted to relaxed states

• Distinct changes in epigenetic states, primarily gain of histone H3 lysine 36 dimethylation (H3K36me2)

• Loss of repressive H3 lysine 27 trimethylation (H3K27me3)

• Unlocking expression of stem cell genes normally silenced during early development

• Enhanced fitness and self-renewal properties of germinal center B-cells

To investigate these changes using the HIST1H1C (Ab-118) antibody, researchers could:

• Perform co-immunofluorescence with HIST1H1C and markers of chromatin modification

• Correlate HIST1H1C expression with expression of stem cell genes (KLF4, KLF5, MEIS1, PRDM5, MYCN)

• Combine immunofluorescence with DNA FISH to analyze changes in chromatin compartmentalization

How should researchers quantify HIST1H1C immunostaining results?

For robust quantification of HIST1H1C immunostaining:

• Capture multiple representative images (minimum 5-10 fields per sample)

• Use image analysis software to quantify:

  • Signal intensity (mean fluorescence intensity)

  • Nuclear vs. cytoplasmic localization

  • Co-localization with other markers

• Include internal controls within each experiment

• Normalize data to account for batch variations

• Perform statistical analysis appropriate for the experimental design

Statistical approaches should account for the distribution of the data and include appropriate tests for the specific experimental design.

What are the key considerations when interpreting HIST1H1C staining patterns in relation to epigenetic modifications?

When analyzing HIST1H1C staining patterns alongside epigenetic markers:

• Remember that HIST1H1C mutations in lymphoma lead to specific patterns of epigenetic alterations:

  • Increased H3K36me1/2 (but not H3K36me3)

  • Decreased H3K27me2/3

  • Little change in other histone modifications

• Consider sequential or dual immunofluorescence to analyze co-occurrence of HIST1H1C with these modifications

• Be aware that HIST1H1C deficiency primarily affects:

  • Developmental PRC2 targets

  • Genes associated with stem cell signatures

  • Genes involved in chromatin compartment B-to-A shifts

• Interpret results in the context of cellular heterogeneity, as subpopulations within samples may show distinct patterns

• Consider that chromatin changes may occur without changes in total HIST1H1C levels, reflecting altered functionality rather than expression

These analytical frameworks provide researchers with robust approaches to interpreting HIST1H1C antibody data in the context of epigenetic modifications and chromatin structure.