DET1 Antibody

What is the DET1 protein and what cellular processes does it regulate?

DET1 functions as an atypical DAMAGED DNA BINDING PROTEIN 1 (DDB1)-CULLIN4 (CUL4) Associated Factor (DCAF) that works with the small DDA1 (DET1-DDB1-Associated 1) protein to provide specificity to E3 CUL4-RING ubiquitin ligases (CRL4) . In plants, DET1 is essential for regulating histone H2B monoubiquitination (H2Bub) across most genes by controlling the stability of a deubiquitination module (DUBm) . This nuclear-localized protein determines cell type-specific expression of light-regulated promoters and represses photomorphogenesis in darkness .

In humans, DET1 controls the stability of cell proliferation factors including the Cdt1 DNA replication-licensing factor and the proto-oncogenic transcription factor c-Jun . Recent research has also shown that DET1 interacts directly with transcription factors CCA1 and LHY1 to regulate the plant circadian clock .

What are the key characteristics of commercially available DET1 antibodies?

Based on available research resources, DET1 antibodies are typically:

When selecting a DET1 antibody, researchers should consider the target regions recognized by different antibodies. Available options include antibodies targeting amino acids 5-190, 15-240, 161-210, or N-terminal regions, which may recognize different functional domains of DET1 .

What are the optimal protocols for using DET1 antibodies in Western blotting?

For optimal results in Western blotting with DET1 antibodies, follow these methodological steps:

• Sample preparation: Lyse cells or tissues in RIPA buffer containing protease inhibitors. For plant tissues, grind in liquid nitrogen before adding buffer to prevent protein degradation.

• Protein quantification: Use Bradford or BCA assays to ensure equal loading (typically 20-50 μg total protein).

• Gel electrophoresis: Separate proteins on 10-12% SDS-PAGE gels, as DET1 has a molecular weight of approximately 55-60 kDa.

• Transfer: Use PVDF membrane for better protein retention and lower background.

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

• Primary antibody incubation: Dilute DET1 antibody at 1:500-1:2000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash membrane 3-4 times with TBST, 5-10 minutes each wash.

• Secondary antibody: Use anti-rabbit HRP-conjugated secondary antibody at 1:5000-1:10000 dilution for 1 hour at room temperature.

• Detection: Visualize using ECL reagents with appropriate exposure times.

For detecting interactions between DET1 and other proteins such as PIFs (Phytochrome Interacting Factors), co-immunoprecipitation followed by Western blotting has proven effective in research settings .

How should DET1 antibodies be stored and handled to maintain optimal reactivity?

Proper storage and handling of DET1 antibodies is critical for maintaining their reactivity and extending shelf life:

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

• Working aliquots: To prevent repeated freeze-thaw cycles, prepare small working aliquots (10-20 μl) upon receipt .

• Buffer conditions: Most DET1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.4 .

• Thawing protocol: Thaw antibodies on ice or at 4°C rather than room temperature.

• Contamination prevention: Use clean pipette tips and sterile technique when handling antibody solutions.

• Transport: When removing from storage, transport on ice and return to -20°C promptly after use.

• Expiration tracking: Document date of receipt and track freeze-thaw cycles to monitor potential degradation.

Note that sodium azide, commonly used as a preservative in antibody solutions, is toxic and should be handled with appropriate precautions .

What species reactivity can I expect from commercially available DET1 antibodies?

When studying DET1 in plant systems, particularly in Arabidopsis where much of the foundational DET1 research has been conducted , antibody selection requires careful consideration of epitope conservation. For cross-species applications, validate antibody specificity using positive and negative controls relevant to your experimental system.

How can DET1 antibodies be utilized to study protein-protein interactions in the ubiquitination pathway?

Studying DET1's role in the ubiquitination machinery requires sophisticated methodological approaches:

• Co-immunoprecipitation (Co-IP): DET1 antibodies can effectively pull down DET1 and its interacting partners. Research has successfully demonstrated Co-IP between DET1 and transcription factors like PIFs in plants . The protocol typically involves:

  • Cross-linking proteins (optional, using 1% formaldehyde)

  • Cell/tissue lysis in non-denaturing buffer

  • Pre-clearing lysate with protein A/G beads

  • Incubating with DET1 antibody (5-10 μg per mg of protein)

  • Capturing complexes with protein A/G beads

  • Washing and eluting for downstream analysis

• Bimolecular Fluorescence Complementation (BiFC): This approach has been used to visualize DET1 interactions with PIFs in vivo . The technique involves:

  • Fusing DET1 and potential interacting partners to complementary fragments of a fluorescent protein

  • Co-expressing constructs in appropriate cell types

  • Monitoring reconstitution of fluorescence when proteins interact

  • Quantifying signal intensity to assess interaction strength

• Proximity-dependent biotin identification (BioID): For identifying novel DET1 interactors in the ubiquitination pathway:

  • Generate fusion proteins of DET1 with a biotin ligase

  • Express in target cells/tissues

  • Proteins in proximity to DET1 become biotinylated

  • Capture biotinylated proteins with streptavidin

  • Identify by mass spectrometry

These methodologies have revealed that DET1 interacts with multiple components of the ubiquitination machinery and various transcription factors, highlighting its central role in protein degradation pathways .

What approaches help resolve discrepancies in results when using different DET1 antibodies?

When different DET1 antibodies yield contradictory results, consider these methodological approaches:

• Epitope mapping analysis:

  • Compare recognition sites of different antibodies (N-terminal, middle region, C-terminal)

  • Consider potential post-translational modifications that might affect epitope accessibility

  • Test antibodies on recombinant DET1 fragments to confirm epitope recognition

• Validation with genetic approaches:

  • Use DET1 knockdown/knockout systems as negative controls

  • Complement with overexpression systems as positive controls

  • Compare antibody signals in wild-type and det1 mutant backgrounds

• Multiple detection methods:

  • Combine immunoblotting with immunofluorescence

  • Validate with mass spectrometry-based approaches

  • Use recombinant tagged DET1 for parallel detection

• Cross-validation protocol:

  Step	Procedure	Purpose
  1	Test multiple antibodies on the same samples	Identify discrepancies
  2	Perform peptide competition assays	Confirm specificity
  3	Use different lysate preparation methods	Assess epitope accessibility
  4	Compare results in multiple cell/tissue types	Evaluate context-dependent differences
  5	Correlate protein detection with mRNA expression	Validate biological relevance

• Consider protein complexes: DET1 functions in multiple protein complexes, including those with DDB1 and CUL4 . Different antibodies may have variable access to DET1 epitopes depending on complex formation.

How can DET1 antibodies help elucidate the role of DET1 in histone modification pathways?

DET1 plays a critical role in regulating histone H2B monoubiquitination (H2Bub) . To investigate this function:

• Chromatin Immunoprecipitation (ChIP):

  • Fix cells/tissues with formaldehyde (1%, 10 minutes)

  • Fragment chromatin (sonication to 200-500 bp fragments)

  • Immunoprecipitate with DET1 antibodies

  • Analyze associated DNA by qPCR or sequencing

  • This approach identifies genomic regions where DET1 is acting

• Sequential ChIP (ChIP-reChIP):

  • Perform initial ChIP with DET1 antibody

  • Elute complexes under non-denaturing conditions

  • Perform second ChIP with antibodies against histone modifications (H2Bub)

  • This confirms co-occupancy of DET1 and specific histone marks

• ChIP-Western analysis:

  • Perform ChIP with DET1 antibody

  • Instead of analyzing DNA, separate proteins by SDS-PAGE

  • Probe membranes for histone modifications or chromatin factors

  • This reveals what proteins associate with DET1 on chromatin

Research has shown that in Arabidopsis, DET1 controls the stability of a deubiquitination module (DUBm) comprising three proteins (SGF11, ENY2, and UBP22) . Unlike yeast and metazoan DUB modules that associate with the large SAGA complex, the Arabidopsis DUBm appears to act independently as a major H2Bub deubiquitinase activity .

What methodologies enable the study of DET1 interactions with transcription factors like PIFs?

To study DET1's interactions with transcription factors such as Phytochrome Interacting Factors (PIFs):

• Yeast two-hybrid (Y2H) screening:

  • Research has shown that a DET1 fragment (amino acids 26-87) interacts with multiple PIF proteins (PIF1, PIF3, PIF4, PIF5, PIF6, and PIF7)

  • Domain mapping experiments revealed that the APB (active phyB binding region) domain of PIF3 is essential for interaction with DET1

  • Y2H can identify which domains of DET1 and PIFs are required for interaction

• In vitro pull-down assays:

  • Express recombinant tagged DET1 (e.g., His-DET1)

  • Immobilize on appropriate resin

  • Incubate with labeled PIF proteins

  • Wash and detect bound proteins

  • This approach has confirmed direct interactions between His-DET1 and GST-PIF proteins

• In vivo validation:

  • Co-immunoprecipitation has shown that endogenous DET1 proteins can be pulled down by Myc-tagged PIF1 and Myc-tagged PIF3

  • BiFC assays have visualized interactions between DET1 and PIF1, PIF3, PIF4, and PIF5 in living cells

• Functional correlation:

  • qRT-PCR analysis can determine whether DET1 affects transcription factor expression levels

  • For example, PIF3 transcript levels were significantly lower in det1-1 mutants than in wild-type plants

  • This approach helps distinguish between effects on protein stability versus transcriptional regulation

These methodologies have collectively established that DET1 interacts with multiple PIF proteins and positively regulates their abundance, contributing to the repression of photomorphogenesis in darkness .

What advanced techniques can be employed to study DET1 dynamics in the ubiquitination pathway?

Recent research on DET1 dynamics in ubiquitination requires sophisticated methodological approaches:

• Real-time ubiquitination assays:

  • Reconstitute CRL4DET1 complex components in vitro

  • Use fluorescently labeled ubiquitin to monitor transfer kinetics

  • Measure effects of DET1 mutations on ubiquitination efficiency

  • Recent biochemical assays suggest E2 ubiquitin-conjugating enzymes act as recruitment factors to bring COP1 to DET1 for more effective substrate ubiquitination

• Protein interaction mapping:

  • Structure-guided mutagenesis has identified critical residues in the DET1-COP1 interface

  • For example, mutations at Glu511 completely abrogated DET1 retention, while mutation at Glu513 did not affect binding

  • Reciprocal mutation on the DET1 side (Y490D) decreased binding to wild-type COP1

  • This approach reveals mechanistic details of the ubiquitination complex assembly

• Advanced microscopy techniques:

  • Fluorescence Resonance Energy Transfer (FRET) to monitor protein-protein interactions in real-time

  • Fluorescence Recovery After Photobleaching (FRAP) to assess DET1 mobility in different cellular compartments

  • Single-molecule tracking to observe DET1 dynamics at individual complex level

• Targeted protein degradation analysis:

  • Identify DET1 substrate degradation kinetics using cycloheximide chase assays

  • Compare degradation profiles in wild-type versus mutant backgrounds

  • Correlate with ubiquitination status using ubiquitin-specific antibodies

These advanced techniques reveal that DET1 functions through dynamic interactions with multiple proteins in the ubiquitination pathway, providing substrate specificity while facilitating efficient ubiquitin transfer .