Phospho-CCNH (T315) Antibody

Basic Research Information

• What is Cyclin H and why is the phosphorylation at T315 significant?

  Cyclin H (CCNH) belongs to the highly conserved cyclin family, serving as a regulator of CDK kinases. It forms a complex with CDK7 and MAT1 to create the CDK-activating kinase (CAK) enzymatic complex . This complex performs two critical functions:

  • Activates cyclin-associated kinases CDK1, CDK2, CDK4, and CDK6 through threonine phosphorylation

  • When complexed with core-TFIIH basal transcription factor, activates RNA polymerase II by phosphorylating the C-terminal domain of its large subunit (POLR2A)

  Phosphorylation at threonine 315 (T315) may regulate these interactions, potentially influencing both cell cycle progression and transcriptional activity. Unlike many cyclins that show periodic expression, Cyclin H maintains constant expression throughout the cell cycle .

• What applications are Phospho-CCNH (T315) antibodies suitable for?

  Based on validation data from multiple suppliers, Phospho-CCNH (T315) antibodies are appropriate for:

  Application	Recommended Dilution	Notes
  Western blot (WB)	1:500-1:2000	Detects ~37 kDa band in human and rodent samples
  Immunohistochemistry (IHC)	1:100-1:300	Works on formalin-fixed paraffin-embedded sections
  ELISA	1:10000	High sensitivity for quantitative detection

  The antibody has been validated using multiple cell lines, including HeLa, H1688, and various tissue lysates from human, mouse, and rat sources .

• What is the molecular basis for antibody recognition of phosphorylated T315?

  Phospho-specific antibodies like Phospho-CCNH (T315) are generated using synthetic phosphopeptides that mimic the region surrounding the phosphorylation site . The antiserum for Phospho-CCNH (T315) antibodies is typically produced against a synthesized peptide derived from human Cyclin H spanning approximately amino acids 274-323, with phosphorylation at T315 . This approach allows the antibody to specifically recognize the conformational change and additional negative charge introduced by the phosphate group, distinguishing the phosphorylated form from the non-phosphorylated version of the protein .

Methodology and Technical Considerations

• How should researchers optimize Western blot protocols for Phospho-CCNH (T315) antibody?

  For optimal Western blot results with Phospho-CCNH (T315) antibody:

  • Sample preparation:

    • Use fresh samples when possible

    • Add phosphatase inhibitors to lysis buffer to preserve phosphorylation status

    • Denature samples at appropriate temperature (typically 95°C for 5 minutes)

  • Protocol optimization:

    • Start with manufacturer-recommended dilution (typically 1:500-1:2000)

    • Use 5% BSA instead of milk for blocking and antibody dilution to prevent non-specific binding

    • Include longer incubation times (overnight at 4°C) for primary antibody

    • Add additional washing steps to reduce background

  • Expected result: A specific band at approximately 37 kDa corresponding to phosphorylated Cyclin H

• What controls should be included when working with Phospho-CCNH (T315) antibody?

  Proper experimental controls are essential for phospho-specific antibody work:

  • Positive control: Cell lysate known to contain phosphorylated CCNH (e.g., HeLa cells)

  • Negative controls:

    • Lambda phosphatase-treated samples (dephosphorylated)

    • Non-phosphorylated peptide competition assay

    • Immunogen peptide blocking (pre-absorption with the phosphorylated peptide should abolish signal)

  • Validation controls:

    • Total CCNH antibody (non-phospho-specific) to confirm protein expression

    • Loading control (β-actin, GAPDH) to ensure equal sample loading

  Abnova's validation data shows clear elimination of signal when their antibody is pre-absorbed by the immunogen peptide, confirming specificity .

• What are the best storage and handling practices for Phospho-CCNH (T315) antibodies?

  For optimal antibody performance and longevity:

  • Storage recommendations:

    • Store at -20°C for long-term (up to one year)

    • For frequent use, aliquot and store small volumes at 4°C for up to one month

    • Avoid repeated freeze-thaw cycles as they can degrade antibody quality

  • Working solution:

    • Maintain in buffer containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

    • Keep on ice when in use

  • Safety note: These antibodies typically contain sodium azide, which is hazardous and should be handled by trained staff only

Advanced Research Applications

• What are the challenges in detecting phosphorylated proteins like CCNH in flow cytometry?

  While flow cytometry is a powerful technique for analyzing phosphorylated proteins, it presents unique challenges:

  • Fixation and permeabilization considerations:

    • Adequate fixation is critical to prevent phosphatase activity (4% formaldehyde recommended)

    • Methanol-free formaldehyde should be used to prevent premature cell permeabilization

    • Different permeabilization methods (saponin, Triton X-100, methanol) may be required depending on the cellular localization

  • Signal optimization:

    • Phospho-epitopes are often present at low abundance, requiring bright fluorochromes

    • Pair phospho-specific antibodies with brighter fluorophores (e.g., PE) rather than dimmer ones (e.g., FITC)

    • Consider fluorochrome size for intracellular/nuclear target accessibility

  • Technical considerations:

    • Cells should be analyzed at lower flow rates to improve resolution

    • Include FMO (fluorescence minus one) controls to distinguish positive from negative signals

    • Block Fc receptors to prevent non-specific binding, especially in monocytes

• How can researchers validate the specificity of Phospho-CCNH (T315) antibody?

  Rigorous validation ensures reliable experimental results:

  • Peptide competition assay:

    • Pre-incubate antibody with phosphorylated peptide (should eliminate signal)

    • Pre-incubate with non-phosphorylated peptide (should not affect signal)

  • Phosphatase treatment:

    • Treat half of your sample with lambda phosphatase

    • Signal should disappear in treated samples only

  • Genetic approaches:

    • Use siRNA/shRNA to knock down CCNH expression

    • Create phospho-mutant (T315A) that cannot be phosphorylated

    • Both approaches should eliminate specific signal

  • Cross-reactivity assessment:

    • Test on multiple cell types with known CCNH expression patterns

    • Evaluate reactivity across species (human, mouse, rat)

• What is the relationship between CCNH phosphorylation and cancer research?

  Research indicates connections between cyclin regulation, phosphorylation states, and cancer development:

  • T315 compounds and cancer therapy:

    • Small molecule T315 has been identified as a novel anticancer drug

    • T315 promotes Casitas B-lineage lymphoma (CBL) activity and triggers EGFR degradation

    • Effective against EGFR-TKI-resistant lung adenocarcinoma cells

  • CCNH in cancer contexts:

    • Cyclin H expression has been detected in various cancer cell lines including HeLa, A431, and K-562

    • Immunohistochemistry shows expression in human lung carcinoma tissue

  While direct links between T315 phosphorylation of CCNH and cancer have not been fully established, the coincidence of terminology (T315 as both a phosphorylation site and a small molecule inhibitor) warrants further investigation into potential mechanistic relationships.

• How does phosphorylation analysis of CCNH compare methodologically to other phospho-protein analysis techniques?

  Phospho-protein analysis requires consideration of various methodological approaches:

  • Western blot vs. flow cytometry:

    • Western blot provides molecular weight confirmation but averages across cell populations

    • Flow cytometry offers single-cell resolution but requires careful optimization for intracellular targets

  • Comparison to STAT protein phosphorylation analysis:

    • Similar to STAT proteins, phosphorylation can occur at both serine and tyrosine residues

    • STAT proteins show distinct "low" and "high" phosphorylation states detectable by flow cytometry

    • Such distinct states have not yet been documented for CCNH phosphorylation

  • Mass spectrometry approaches:

    • Enables quantitative measurement of multiple phosphorylation sites simultaneously

    • Higher sensitivity than antibody-based methods for detecting low-abundance modifications

    • Requires specialized equipment and expertise not available in all laboratories

• What are the recommended troubleshooting approaches for weak or no signal when using Phospho-CCNH (T315) antibody?

  When encountering detection issues:

  Problem	Possible Causes	Recommended Solutions
  No signal	Insufficient target protein	Confirm CCNH expression in your samples
  	Target not phosphorylated	Verify conditions that induce T315 phosphorylation
  	Improper antibody storage	Ensure antibody hasn't been repeatedly freeze-thawed
  Weak signal	Suboptimal antibody dilution	Try more concentrated primary antibody solution
  	Inadequate incubation time	Extend primary antibody incubation (overnight at 4°C)
  	Inefficient transfer (WB)	Optimize transfer conditions for ~37 kDa proteins
  High background	Insufficient blocking	Increase blocking time/concentration
  	Inadequate washing	Add more wash steps between antibody incubations
  	Cross-reactivity	Use blocking peptides to confirm specificity

  If standard troubleshooting fails, consider using a different detection method or antibody clone .

• What recent advances in phospho-antibody technology might improve detection of CCNH phosphorylation?

  Emerging technologies for phospho-specific detection include:

  • Liposome-based vaccines for generating highly specific phospho-antibodies:

    • Incorporating phosphorylated peptides onto liposomal surfaces has proven both safe and efficacious

    • This approach has been successful for generating antibodies against phospho-Tau

    • Similar methods could potentially enhance specificity of phospho-CCNH antibodies

  • Simultaneous detection of multiple phosphorylation sites:

    • Flow cytometry panels can now assess up to eight phosphorylated residues simultaneously

    • This could enable correlation analysis between CCNH T315 phosphorylation and other signaling events

  • Enhanced validation approaches:

    • Systematic analysis of phospho-antibody cross-reactivity using peptide arrays

    • CRISPR-based knockout validation to confirm true specificity in cellular contexts