HCFC1 Antibody

What are the key considerations when selecting an HCFC1 antibody for my research?

When selecting an HCFC1 antibody, several critical factors should be considered:

• Target epitope location: HCFC1 is proteolytically cleaved, resulting in N-terminal and C-terminal fragments. Choose antibodies targeting specific regions based on your research goals:

  • N-terminal antibodies (e.g., ab137618) detect the N-terminal fragments

  • C-terminal antibodies (e.g., #50708) detect the C-terminal fragments

  • Full-length antibodies may detect both fragments

• Species reactivity: Verify cross-reactivity with your experimental model. Most commercial HCFC1 antibodies react with:

  • Human (all antibodies in the search results)

  • Mouse (AF6254, CAB16871, 28569-1-AP, 19358-1-AP, #50708)

  • Rat (CAB16871, 28569-1-AP, 19358-1-AP, #50708)

• Application compatibility: Ensure the antibody is validated for your specific application:

• Clonality: Consider whether monoclonal or polyclonal antibodies better suit your experimental needs .

How can I validate the specificity of my HCFC1 antibody?

Comprehensive validation should include:

• Positive control selection: Use cell lines known to express HCFC1, such as:

  • HeLa human cervical epithelial carcinoma cells

  • Daudi human Burkitt's lymphoma cells

  • HEK-293 cells

  • A431 cells

  • Jurkat cells

• Multiple detection methods: Confirm specificity using:

  • Western blot: Look for bands at approximately 300 kDa (full-length) and 100-175 kDa (proteolytic fragments)

  • Immunofluorescence: Verify nuclear localization pattern

  • Knockdown/knockout validation: Use siRNA or CRISPR to reduce HCFC1 expression and confirm signal reduction

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to demonstrate signal specificity .

What is the optimal sample preparation protocol for detecting HCFC1 in Western blots?

For optimal HCFC1 detection in Western blots:

• Lysis buffer selection: Use buffers containing protease inhibitors to prevent further degradation of HCFC1 fragments

• Gel selection: Use low percentage (6-8%) gels for better resolution of high molecular weight proteins

• Running conditions:

  • Use reducing conditions as demonstrated in validated protocols

  • When using PVDF membrane, follow specific protocols like Immunoblot Buffer Group 1 (R&D Systems)

• Antibody dilution:

  • AF6254: 1 μg/mL

  • 28569-1-AP: 1:1000-1:8000

  • 19358-1-AP: 1:500-1:2000

  • #50708: 1:1000

• Expected band patterns: Be prepared to see multiple bands:

  • Full-length HCFC1: ~300 kDa

  • Proteolytic fragments: ~100-175 kDa

  • Additional processing variants: 100, 125, 130, 145, 260 kDa

What are the recommended protocols for immunohistochemical detection of HCFC1?

For optimal immunohistochemical detection:

• Tissue preparation:

  • Use immersion-fixed paraffin-embedded sections

  • For antigen retrieval, use TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0)

• Antibody incubation:

  • AF6254: 10 μg/mL overnight at 4°C

  • 28569-1-AP: 1:1000-1:4000 dilution

  • Counterstain with hematoxylin for contrast

• Detection systems:

  • For AF6254 (goat antibody): Anti-Goat HRP-DAB Cell & Tissue Staining Kit (R&D Systems, CTS008)

  • For rabbit antibodies: Appropriate HRP-conjugated secondary antibodies

• Positive control selection:

  • Human colon cancer tissue (validated for AF6254)

  • Mouse liver tissue, human lung cancer tissue, human urothelial carcinoma tissue (validated for 28569-1-AP)

Why am I detecting multiple bands of varying molecular weights when using HCFC1 antibodies?

The detection of multiple bands is expected and biologically relevant when working with HCFC1:

• Biological explanation:

  • HCFC1 is synthesized as a 2035-amino-acid precursor (calculated MW: 209 kDa)

  • HCFC1 undergoes proteolytic cleavage at the PRO repeats by O-GlcNAc transferase (OGT)

  • This produces a heterodimeric complex of HCFC1N and HCFC1C subunits

  • These fragments typically appear between 100-175 kDa

• Observed molecular weight variations:

  • Full-length protein: Observed at 230-300 kDa (higher than calculated due to post-translational modifications)

  • N-terminal fragments: Typically 100-150 kDa

  • C-terminal fragments: Typically 100-175 kDa

  • Additional bands may represent cell type-specific processing variants

• Technical considerations:

  • Use appropriate molecular weight markers that cover the full range (50-300 kDa)

  • Run control samples (HeLa, Daudi) alongside experimental samples for comparison

  • Different antibodies may preferentially detect certain fragments based on their epitope

What are common troubleshooting approaches for weak or absent HCFC1 signal in immunofluorescence experiments?

When encountering weak or absent signals:

• Fixation optimization:

  • HCFC1 detection works best with immersion-fixed samples

  • Overfixation can mask epitopes; consider titrating fixation times

• Antibody concentration adjustment:

  • For AF6254: Try 5-15 μg/mL range

  • For ab137618 and 28569-1-AP: Test 1:50-1:500 dilution range

  • Increase incubation time (3 hours at room temperature or overnight at 4°C)

• Signal amplification strategies:

  • For IF/ICC: Use high-sensitivity detection systems like NorthernLights™ 557-conjugated secondary antibodies

  • Counterstain with DAPI to confirm nuclear localization

  • Consider tyramide signal amplification for low abundance targets

• Background reduction:

  • Increase blocking time or concentration

  • Include 0.1-0.3% Triton X-100 for better nuclear penetration

  • Pre-absorb secondary antibodies with tissue powder from the same species

How can HCFC1 antibodies be used to study its role in transcriptional regulation complexes?

HCFC1 antibodies can provide insights into transcriptional regulation through:

• Chromatin immunoprecipitation (ChIP):

  • Use highly specific antibodies like 28569-1-AP or #50708 for immunoprecipitation

  • Follow with sequencing (ChIP-seq) to identify genome-wide binding sites

  • Analyze enrichment at CpG islands, as HCFC1 is a common component of active human CpG-island promoters

• Co-immunoprecipitation (Co-IP):

  • Identify protein interaction partners in different cellular contexts

  • Study HCFC1 interactions with:

    • Set1/Ash2 histone H3 'Lys-4' methyltransferase (H3K4me)

    • Sin3 histone deacetylase (HDAC) complexes

    • THAP1/THAP3-HCFC1-OGT complex

    • E2F1 and other transcription factors

• Proximity ligation assay (PLA):

  • Visualize in situ protein-protein interactions

  • Study the spatial relationship between HCFC1 and its binding partners

  • Detect transient interactions during cell cycle progression

What considerations are important when using HCFC1 antibodies in cancer research studies?

When studying HCFC1 in cancer:

• Tissue-specific expression patterns:

  • HCFC1 shows differential expression across cancer types

  • Validated in colon cancer tissue, hepatocellular carcinoma, lung cancer and urothelial carcinoma

  • Consider matched normal/tumor tissue pairs for comparative analysis

• Correlation with clinical parameters:

  • Study associations between HCFC1 expression and:

    • Cell cycle progression markers

    • Immune infiltration profiles

    • Patient survival data

  • Upregulation of HCFC1 expression promotes hepatocellular carcinoma progression by inhibiting cell cycle arrest

• Functional studies:

  • Combine antibody-based detection with genetic manipulation (knockdown/overexpression)

  • Evaluate changes in:

    • Cell proliferation

    • Migration

    • Cell death

    • Mitochondrial biogenesis

How can HCFC1 antibodies be used to study its role in herpes simplex virus (HSV) infection?

HCFC1 was first identified in HSV transcription studies. To investigate this relationship:

• Infection models:

  • Use human cell lines susceptible to HSV infection (e.g., HeLa, Vero)

  • Compare HCFC1 localization and processing before and after infection

  • Track temporal changes during the viral life cycle

• Protein complex analysis:

  • Study the multiprotein-DNA complex formation between HCFC1, viral transactivator protein VP16, and POU2F1

  • Use co-immunoprecipitation with HCFC1 antibodies to pull down viral interaction partners

  • Apply chromatin immunoprecipitation to identify viral immediate early gene promoters

• Functional assessment:

  • Use HCFC1 antibodies in conjunction with viral protein antibodies to track co-localization

  • Evaluate how HCFC1 knockdown affects viral transcription and replication

  • Study post-translational modifications of HCFC1 during infection

What methodological approaches are recommended for studying HCFC1 proteolytic processing?

To investigate HCFC1 processing:

• Fragment-specific detection:

  • Use N-terminal-specific antibodies (e.g., ab137618, CAB16871) to detect N-terminal fragments

  • Use C-terminal-specific antibodies (e.g., #50708) to detect C-terminal fragments

  • Compare patterns across different cell types and conditions

• Processing inhibition studies:

  • Investigate the role of O-GlcNAc transferase (OGT) in HCFC1 cleavage

  • Apply OGT inhibitors and monitor changes in HCFC1 processing

  • Use HCFC1 antibodies to detect shifts in the ratio of full-length to processed forms

• Mass spectrometry validation:

  • Immunoprecipitate HCFC1 using validated antibodies (28569-1-AP, #50708)

  • Analyze by mass spectrometry to identify precise cleavage sites

  • Map post-translational modifications that may regulate processing

• Mutation analysis:

  • Study how mutations in HCFC1 affect its processing and function

  • Particularly relevant for X-linked recessive diseases associated with HCFC1 mutations

  • Compare wild-type and mutant protein processing patterns using appropriate antibodies

How should HCFC1 antibodies be applied in studies of chromatin modification and epigenetic regulation?

For chromatin and epigenetic studies:

• Sequential ChIP approaches:

  • Perform ChIP with HCFC1 antibodies followed by re-ChIP with antibodies against histone modifications

  • Identify genomic regions where HCFC1 coincides with specific epigenetic marks

  • Focus on the association with the Set1/Ash2 histone H3 'Lys-4' methyltransferase and Sin3 histone deacetylase complexes

• Co-localization studies:

  • Combine HCFC1 immunofluorescence with detection of:

    • Histone modifications (H3K4me3, H3K27ac)

    • Chromatin remodelers

    • Transcriptional machinery components

  • Use high-resolution microscopy to visualize nuclear co-localization patterns

• NSL complex participation:

  • Investigate HCFC1's role in the NSL complex for nucleosomal histone H4 acetylation

  • Study HCFC1 recruitment to E2F1-responsive promoters facilitating G1 to S phase transition

  • Correlate HCFC1 localization with transcriptional activation markers