DEGP7 Antibody

What is DEGP7 protease and what cellular functions does it serve?

DEGP7 (Protease Do-like 7, EC=3.4.21) is a serine/cysteine protease primarily identified in plant systems, particularly Arabidopsis thaliana. It belongs to the DEG/HtrA family of ATP-independent serine proteases that generally function in protein quality control mechanisms. DEGP7 plays a crucial role in the photoinhibition process, which involves protective mechanisms against light-induced damage in photosynthetic systems .

The protein is predominantly localized in plant nuclei, as evidenced by its detection in enriched nuclear fractions but not in total cell extracts. This localization pattern suggests specialized functions potentially related to nuclear protein quality control or signaling pathways connecting nuclear processes with photosynthetic activity regulation. Recent research indicates DEGP7 may be particularly important during stress responses, serving as part of the cellular machinery that maintains protein homeostasis under adverse conditions.

What are the key specifications of commercially available anti-DEGP7 antibodies?

The following table summarizes the key specifications of a typical anti-DEGP7 antibody used in plant research:

This antibody has been specifically developed to recognize epitopes on the DEGP7 protein in Arabidopsis thaliana and has been validated in Brassica oleracea as well .

What are the optimal handling and storage conditions for DEGP7 antibodies?

For maintaining optimal activity of anti-DEGP7 antibodies, follow these methodological guidelines:

• Storage conditions: Store lyophilized antibody at -20°C in a non-frost-free freezer to avoid temperature fluctuations. After reconstitution, continue storage at -20°C .

• Reconstitution protocol:

  • Add 200 μl of sterile water to the lyophilized antibody

  • Gently mix to ensure complete dissolution

  • Allow to stand at room temperature for 5 minutes before use

  • For long-term storage, prepare multiple small aliquots to minimize freeze-thaw cycles

• Sample handling: Always centrifuge tubes briefly before opening to collect any material that may adhere to the cap or sides of the tube, preventing potential loss of antibody .

• Freeze-thaw considerations: Limit freeze-thaw cycles to preserve antibody activity. Each cycle can potentially reduce activity by 10-15%. Date each aliquot and track usage to maintain experimental consistency.

• Working solution preparation: Prepare fresh dilutions on the day of experiment. Dilute in appropriate buffer containing 0.1-0.5% BSA to minimize non-specific binding.

Which experimental applications are DEGP7 antibodies suitable for?

Based on current validation data, anti-DEGP7 antibodies are most effectively used in the following applications:

• Western blot analysis (recommended dilution 1:500): The primary validated application, particularly effective when working with enriched nuclear fractions. When performing Western blots:

  • Use 10-20 μg of enriched nuclear protein per lane

  • Include molecular weight markers to confirm the expected 119.8 kDa band

  • Employ enhanced chemiluminescence detection for optimal sensitivity

• Immunoprecipitation: While not explicitly validated in the search results, immunoprecipitation protocols may be developed using standard approaches:

  • Pre-clear lysates to reduce background

  • Use 2-5 μg antibody per 500 μg of nuclear protein extract

  • Incubate overnight at 4°C with gentle rotation

  • Validate results with Western blot analysis

• Immunolocalization: For subcellular localization studies, consider:

  • Optimization of fixation conditions to preserve both antigenicity and cellular architecture

  • Including appropriate controls (see section 2.8)

  • Using confocal microscopy to precisely determine nuclear localization

Note that the antibody does not provide adequate signal with total cell extracts, indicating a requirement for sample enrichment strategies .

How should researchers validate DEGP7 antibody specificity in their experimental systems?

Methodological approach to antibody validation:

• Genetic validation: Utilize DEGP7 knockout or knockdown plant lines as negative controls. Absence of signal in these lines provides strong evidence of specificity.

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (10-100 fold molar excess)

  • Run parallel Western blots with competed and non-competed antibody

  • Specific signals should be significantly reduced or eliminated in the competed sample

• Cross-species validation: If working with non-model species, test antibody reactivity against purified recombinant DEGP7 proteins from the species of interest.

• Molecular weight confirmation: Verify that the detected band corresponds to the predicted molecular weight of DEGP7 (119.8 kDa in Arabidopsis thaliana) .

• Signal specificity assessment: Confirm that the antibody produces stronger signals in nuclear-enriched fractions compared to total cell extracts, consistent with the known localization pattern of DEGP7.

Why does anti-DEGP7 antibody produce signals in enriched nuclei fractions but not in total cell extracts?

This phenomenon likely results from a combination of factors:

• Relative abundance considerations: DEGP7 may constitute a small percentage of total cellular protein, falling below detection thresholds in whole-cell extracts. Nuclear enrichment effectively concentrates the target protein to detectable levels .

• Subcellular compartmentalization: The predominant nuclear localization of DEGP7 means it represents a much higher proportion of protein in nuclear fractions compared to total cell extracts.

• Epitope accessibility: Total cell extraction protocols may employ conditions that alter the DEGP7 epitope structure or accessibility. Nuclear enrichment protocols may better preserve the native conformation necessary for antibody recognition.

• Experimental methodology implications: For successful detection:

  • Use nuclear enrichment protocols that maintain protein integrity

  • Verify enrichment efficiency through nuclear marker proteins

  • Ensure extraction buffers include appropriate protease inhibitors

  • Optimize protein loading (typically 15-20 μg for nuclear extracts)

• Biological significance: This localization pattern suggests DEGP7 may have specialized nuclear functions distinct from classical chloroplast-localized photoinhibition processes.

What molecular mechanisms underlie DEGP7's role in the photoinhibition process?

The involvement of DEGP7 in photoinhibition appears to operate through several potential mechanisms:

• Protein quality control: As a serine/cysteine protease, DEGP7 likely participates in the detection and degradation of damaged proteins resulting from light-induced oxidative stress .

• Nuclear-chloroplast signaling: Given its nuclear localization, DEGP7 may function in retrograde signaling pathways that coordinate nuclear gene expression with chloroplast status during photoinhibition.

• Stress response regulation: DEGP7 may process specific transcription factors or other regulatory proteins involved in activating stress response genes.

• Proteolytic cascade involvement: DEGP7 could function as part of a larger proteolytic network, with its activity triggering or modulating other proteases in response to photoinhibition signals.

• Target specificity: Unlike other DEG family proteases that primarily target photosystem proteins directly, DEGP7's nuclear localization suggests it may regulate factors controlling expression of photosynthetic machinery components.

Further research employing chromatin immunoprecipitation (ChIP) combined with nuclear proteomics could help identify DEGP7's specific nuclear targets during photoinhibition response.

What is known about the oligomerization principles of plant DEGP7 protease?

Research by Schuhmann et al. (2011) has revealed novel insights into DEGP7 oligomerization:

• Unique assembly mechanism: DEGP7 employs a previously undescribed oligomerization principle based on interactions between degenerated protease domains .

• Structural organization: Unlike other proteases that typically form homogeneous multimers, DEGP7 oligomerization involves interactions between structurally distinct domains within the same protein.

• Functional implications: This unique oligomerization mechanism likely contributes to:

  • Regulated substrate access to active sites

  • Cooperative activity regulation in response to cellular signals

  • Specific subcellular localization patterns

  • Enhanced stability under stress conditions

• Evolutionary perspective: This represents a potentially plant-specific adaptation of the DEG/HtrA family that may facilitate specialized functions in photosynthetic organisms.

• Methodological considerations: When designing experiments to study DEGP7:

  • Consider native versus denaturing conditions carefully

  • Include controls that account for potential oligomeric state changes

  • Evaluate how experimental conditions might affect complex stability

What sample preparation techniques maximize DEGP7 detection efficiency?

Based on the limited detection in total cell extracts, the following methodological approaches are recommended:

• Nuclear enrichment protocol:

  • Homogenize plant tissue in nuclear isolation buffer (20 mM Tris-HCl pH 7.4, 25% glycerol, 2.5 mM MgCl₂, 0.5% Triton X-100)

  • Filter through miracloth to remove debris

  • Layer filtrate onto a 30% percoll cushion and centrifuge (1,000 × g, 5 min)

  • Wash nuclear pellet twice with nuclear isolation buffer

  • Extract nuclear proteins using high-salt buffer (50 mM Tris-HCl pH 7.4, 400 mM NaCl, 1 mM EDTA)

• Protease inhibitor considerations:

  • Include a comprehensive inhibitor cocktail (PMSF, leupeptin, pepstatin, aprotinin)

  • Add fresh inhibitors immediately before sample processing

  • Maintain samples at 4°C throughout preparation

• Buffer optimization:

  • Test multiple extraction buffers with varying ionic strengths

  • Consider non-ionic detergents to improve solubilization

  • Evaluate pH ranges (7.0-8.0) for optimal epitope preservation

• Quantification and loading:

  • Determine protein concentration using Bradford or BCA assay

  • Load 15-20 μg of nuclear protein per lane for optimal detection

  • Include nuclear marker proteins (histone H3) as loading controls

How does DEGP7 structure relate to its function and localization patterns?

While detailed structural information is limited in the search results, we can infer structure-function relationships:

• Domain organization:

  • Contains catalytic serine/cysteine protease domain

  • Likely includes nuclear localization signals

  • Features degenerated protease domains involved in oligomerization

• Localization determinants:

  • Nuclear targeting sequences direct DEGP7 to the nucleus

  • Possible retention mechanisms through interaction with nuclear components

  • Potential for conditional localization depending on cellular state

• Catalytic mechanism:

  • As an EC 3.4.21 enzyme, operates via typical serine protease catalytic triad

  • Substrate specificity likely determined by surface loops and binding pockets

  • Activity may be regulated through conformational changes upon oligomerization

• Evolutionary conservation:

  • Higher conservation expected in catalytic domains across species

  • Greater variability in regulatory regions and localization signals

  • Species-specific adaptations possibly reflecting different photoinhibition mechanisms

What experimental controls are essential when using anti-DEGP7 antibody for immunolocalization?

For rigorous immunolocalization studies, implement these critical controls:

• Negative controls:

  • Omission of primary antibody: Reveals non-specific binding of secondary antibody

  • Pre-immune serum: Indicates background from host animal antibodies

  • DEGP7 knockout/knockdown plant material: Confirms signal specificity

• Epitope competition:

  • Pre-incubate antibody with immunizing peptide

  • Run parallel experiments with competed and non-competed antibody

  • Specific signal should be substantially reduced with competition

• Subcellular markers:

  • Include established nuclear markers (e.g., histone proteins)

  • Co-localize with DNA stains (DAPI)

  • Compare pattern with other known nuclear proteins

• Fixation validations:

  • Test multiple fixation protocols (paraformaldehyde, glutaraldehyde)

  • Compare chemical versus cryo-fixation methods

  • Verify that fixation doesn't alter epitope recognition

• Cross-validation:

  • Confirm localization with orthogonal techniques (e.g., GFP fusions)

  • Correlate immunolocalization with cell fractionation results

  • Consider super-resolution microscopy for precise nuclear sublocalization

What considerations apply when using anti-DEGP7 antibody across different plant species?

When extending DEGP7 research beyond model systems, consider:

• Sequence conservation analysis:

  • Examine DEGP7 sequence homology between target species and Arabidopsis

  • Focus particularly on the epitope region used for antibody generation

  • Higher homology suggests greater likelihood of cross-reactivity

• Validation strategies:

  • Begin with Western blot analysis to confirm recognition at appropriate molecular weight

  • Verify nuclear enrichment pattern is maintained across species

  • Consider generating species-specific antibodies for distant taxa

• Optimization parameters:

  • Test multiple antibody concentrations (typically 2-5× higher for distant species)

  • Modify blocking conditions to reduce background

  • Adjust incubation times and temperatures to enhance specific binding

• Alternative approaches:

  • For distantly related species, consider epitope-tagged DEGP7 expression

  • Employ mass spectrometry to confirm antibody target identity

  • Use complementary techniques like RNA analysis to support protein studies

How can researchers investigate protein-protein interactions involving DEGP7?

To elucidate DEGP7's interaction network:

• Co-immunoprecipitation approach:

  • Use anti-DEGP7 antibody coupled to protein A/G beads

  • Perform IP under gentle conditions to maintain interactions

  • Identify binding partners through mass spectrometry

  • Validate key interactions with reverse co-IP

• Yeast two-hybrid screening:

  • Generate DEGP7 bait constructs, considering full-length and domain-specific versions

  • Screen against plant cDNA libraries

  • Validate positive interactions with alternative methods

  • Assess interaction domains through deletion constructs

• Proximity labeling techniques:

  • Create DEGP7-BioID or DEGP7-APEX2 fusion proteins

  • Express in plant cells and activate labeling

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Distinguish between stable interactions and transient associations

• Gel filtration analysis:

  • Investigate oligomerization states under varying conditions

  • Determine if DEGP7 exists in multiple complexes

  • Examine how stress conditions affect complex formation

  • Study the effect of substrates on complex stability

• Considerations for interaction studies:

  • Focus on nuclear extracts based on known localization

  • Include appropriate controls for specificity

  • Consider light/dark conditions given photoinhibition connection

  • Evaluate interactions under stress versus normal conditions