HIC1 Antibody, Biotin conjugated

What is HIC1 and why is it significant in cancer research?

HIC1 (also known as ZBTB29, ZNF901) is a tumor suppressor gene that encodes the HIC ZBTB transcriptional repressor 1 protein. This gene is frequently hypermethylated and consequently silenced in various cancer types. Research indicates that HIC1 deletion significantly correlates with breast cancer progression and poor prognosis . Gene expression analyses reveal that HIC1 mRNA levels are notably lower in breast cancer tissues compared to normal tissues from the same patients, with particularly reduced expression in triple-negative breast cancer (TNBC) cases . Additionally, HIC1 expression decreases in high-grade breast cancer tissues compared to low-grade tissues, and its deletion correlates with lower relapse-free survival, suggesting its importance as a prognostic marker .

What are the typical biochemical characteristics of HIC1 protein?

HIC1 protein has the following characteristics:

• Calculated molecular weight: 75-76.5 kDa

• Observed molecular weight: 65-70 kDa in Western blot analyses

• Full-length protein contains 714 amino acids

• Contains multiple functional domains including zinc finger domains

What species reactivity can be expected from commercially available HIC1 antibodies?

HIC1 antibodies demonstrate various cross-reactivity profiles depending on the specific product:

Most HIC1 antibodies show strong reactivity with human samples, with varying degrees of cross-reactivity to other mammalian species .

How should researchers validate the specificity of biotin-conjugated HIC1 antibodies?

Validation of biotin-conjugated HIC1 antibodies should follow a multi-step approach:

• Western blot analysis using positive control cell lysates (e.g., Jurkat or NIH/3T3 cells) to confirm detection at the expected molecular weight (65-70 kDa)

• Include appropriate negative controls such as:

  • Isotype control antibodies with matching biotin conjugation

  • Tissues/cells known to have HIC1 deletion or knockdown

• Peptide competition assay using the immunogen peptide (e.g., N-terminal region of human HIC1) to confirm specificity

• For immunohistochemistry applications, compare staining patterns with published literature and validate with multiple antibodies targeting different epitopes

• Perform siRNA knockdown experiments to demonstrate reduction in signal intensity correlating with reduced HIC1 expression

What are the optimal conditions for using biotin-conjugated HIC1 antibodies in Western blotting?

For optimal Western blot results with biotin-conjugated HIC1 antibodies:

• Sample preparation:

  • Use standard cell lysis buffers containing protease inhibitors

  • For nuclear proteins like HIC1, consider nuclear extraction protocols

  • Load 20-50 μg of total protein per lane

• Electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels due to the 65-70 kDa size of HIC1

  • Transfer to PVDF membrane at 100V for 60-90 minutes

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • For direct detection of biotin-conjugated antibodies, use streptavidin-HRP (1:1000-1:5000)

  • For larger signal amplification, use an avidin-biotin complex (ABC) system

  • Recommended antibody dilution ranges: 1:500-1:2000

• Detection:

  • Use enhanced chemiluminescence (ECL) reagents

  • Expose membrane for 30 seconds to 5 minutes, depending on signal strength

How can biotin-conjugated HIC1 antibodies be utilized in proximity labeling experiments?

Biotin-conjugated HIC1 antibodies can be powerful tools in proximity labeling experiments using methodologies like Biotinylation by Antibody Recognition (BAR) :

• In fixed and permeabilized tissue samples:

  • Primary HIC1 antibody is used to target the protein of interest

  • Secondary HRP-conjugated antibody creates free radicals in the presence of hydrogen peroxide and phenol biotin

  • This results in biotinylation of proteins in close proximity to HIC1

• For protein interaction studies:

  • After biotinylation, harsh conditions can be used for reverse cross-linking and protein solubilization

  • Streptavidin-coated beads precipitate biotinylated proteins

  • Precipitated proteins are identified by tandem mass-spectrometry

• For quantitative comparison, Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) can be used to contrast signal from the target of interest with background signal

This method has advantages over traditional co-immunoprecipitation approaches, particularly for detecting interactions involving insoluble proteins that form higher-order structures .

What considerations should be made when using biotin-conjugated HIC1 antibodies in immunohistochemistry?

When using biotin-conjugated HIC1 antibodies for immunohistochemistry:

• Antigen retrieval considerations:

  • For optimal results, use TE buffer pH 9.0

  • Alternative method: citrate buffer pH 6.0

• Endogenous biotin blocking:

  • Tissues may contain endogenous biotin, particularly liver, kidney, and breast tissue

  • Pre-block with avidin/biotin blocking kit

  • Consider using streptavidin-based detection systems that show lower background

• Dilution optimization:

  • Starting dilution recommendation: 1:500-1:2000

  • Sample-dependent, requiring titration for optimal results

• Validation with positive controls:

  • Confirmed positive tissues include rat lung and stomach tissue

• Signal amplification systems:

  • For low-expression samples, consider tyramide signal amplification (TSA)

  • When using TSA, further dilute primary antibody (1:5000-1:10000) to reduce background

How can researchers overcome non-specific binding when using biotin-conjugated HIC1 antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies. Researchers can employ these strategies:

• For Western blotting:

  • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Use alternative blocking agents (5% BSA, commercial blocking buffers)

  • Add 0.1-0.5% Tween-20 to washing buffers

  • Increase washing duration and number of wash steps

  • Pre-absorb antibody with cell/tissue lysate from species not expressing the target

• For immunohistochemistry/immunofluorescence:

  • Use avidin/biotin blocking kit to block endogenous biotin

  • Block with serum from the same species as the secondary antibody

  • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific membrane binding

  • Consider using directly labeled primary antibodies to avoid secondary antibody cross-reactivity

• For immunoprecipitation:

  • Use protein A/G beads pre-cleared with the sample lysate

  • Add non-immune IgG from the same species as the antibody to the lysis buffer

What are the potential artifacts in proximity labeling experiments using biotin-conjugated HIC1 antibodies?

Proximity labeling with biotin-conjugated antibodies can introduce several artifacts:

• Labeling radius considerations:

  • Biotin phenol radicals typically have a labeling radius of ~20-30 nm

  • This may capture proteins that are proximal but not direct interactors

  • Control experiments with known non-interactors at similar distances are recommended

• Time-dependent enrichment:

  • Biotinylation levels increase with biotin exposure duration

  • Saturation typically occurs within 6-24 hours

  • Standardize exposure time across experimental and control conditions

• Expression level artifacts:

  • Overexpression can lead to mislocalization and false interactions

  • Consider using endogenous expression systems or carefully titrated expression levels

• Cell fixation artifacts:

  • Fixation can cause protein crosslinking that doesn't represent native interactions

  • Compare results from different fixation methods

  • Consider complementary live-cell approaches

How can temporal regulation be achieved in biotinylation experiments involving HIC1?

Temporal regulation of biotinylation enables dynamic studies of protein interactions:

• Biotin-dependent activation:

  • BirA* biotin ligase activity is dependent on exogenous biotin

  • Addition of 50 μM biotin to culture medium stimulates promiscuous biotinylation

  • Biotinylation levels increase with biotin exposure duration, reaching saturation within 6-24 hours

• Pulse-chase experimental design:

  • Brief biotin exposure (1-4 hours) allows "pulse" labeling

  • Wash and replace with biotin-free medium for "chase" period

  • This permits tracking interaction dynamics over time

• Inducible expression systems:

  • Use doxycycline-inducible promoters to control expression of biotinylation enzymes

  • Combine with controlled biotin exposure for precise temporal regulation

  • Example: induce expression with doxycycline and add 50 μM biotin for 24 hours

What approaches can be used to study HIC1's role in breast cancer progression?

Based on recent research findings, several methodological approaches can be employed:

• HIC1 deletion studies:

  • Research indicates HIC1-deleted breast cancer cells can directly induce activation of stromal fibroblasts in mammary gland both in vivo and in vitro

  • Co-culture experiments with HIC1-deleted cancer cells and normal stromal fibroblasts

  • Analysis of fibroblast activation markers (α-SMA, FAP, etc.)

• Correlation with clinical outcomes:

  • Expression analysis shows HIC1 mRNA levels are significantly lower in cancer tissues than normal tissues

  • Lower expression in triple-negative breast cancer compared to non-TNBC patients

  • Decreased expression in high-grade tumors compared to low-grade

  • Kaplan-Meier analysis shows HIC1 deletion correlates with lower relapse-free survival

• Mechanistic studies:

  • Investigate transcriptional targets of HIC1 using ChIP-seq with HIC1 antibodies

  • Analyze pathway activation in HIC1-deleted versus HIC1-expressing cells

  • Determine if HIC1 restoration in deleted cells reverses malignant phenotypes

How can biotin-conjugated HIC1 antibodies contribute to single-cell protein interaction studies?

Recent advances in single-cell technologies offer new applications for biotin-conjugated HIC1 antibodies:

• Integration with microfluidic platforms:

  • Capture individual cells in droplets or chambers

  • Perform in situ proximity labeling with biotin-conjugated HIC1 antibodies

  • Combine with single-cell RNA-seq to correlate protein interactions with transcriptional profiles

• Mass cytometry applications:

  • Use biotin-conjugated HIC1 antibodies followed by metal-tagged streptavidin

  • Enables detection of HIC1 protein in CyTOF experiments

  • Combine with other metal-tagged antibodies for multiparameter analysis

• Spatial transcriptomics integration:

  • Apply biotin-conjugated HIC1 antibodies to tissue sections

  • Detect with fluorescent streptavidin conjugates

  • Overlay with spatial transcriptomics data to correlate protein localization with gene expression patterns

What are the considerations for using HIC1 antibodies in studying epigenetic regulation?

HIC1 (Hypermethylated in Cancer 1) has important roles in epigenetic regulation, requiring specific methodological considerations:

• Chromatin immunoprecipitation (ChIP) optimization:

  • For formaldehyde cross-linking, use 1% formaldehyde for 10 minutes at room temperature

  • Sonication conditions: optimize to generate 200-500 bp DNA fragments

  • For biotin-conjugated antibodies, use streptavidin beads for precipitation

  • Input control and IgG controls are essential for accurate data interpretation

• DNA methylation analysis:

  • Combine HIC1 ChIP with bisulfite sequencing to correlate HIC1 binding with DNA methylation status

  • Consider hydroxymethylation (5-hmC) analysis to distinguish from 5-mC

  • Methyl-DIP followed by HIC1 ChIP can reveal methylation-dependent binding patterns

• Histone modification studies:

  • Sequential ChIP (HIC1 followed by histone mark antibodies) can reveal chromatin states at HIC1 binding sites

  • Consider using antibodies against specific histone modifications (H3K27me3, H3K9me3) that may co-occur with HIC1 binding