OFD1 Antibody, HRP conjugated

What is OFD1 protein and why is it important in research?

OFD1 is a protein encoded by the OFD1 gene responsible for Oral-facial-digital type I syndrome, an X-linked dominant disorder characterized by malformations of the oral cavity, face, digits, and cystic kidneys. OFD1 is primarily localized to the centrosome and primary cilium, but research has shown it also localizes to the nucleus . The protein contains two main structural characteristics: coiled-coil (CC) domains that mediate protein-protein interactions and a LisH motif which can act as a dimerization motif . OFD1 is important in research because it represents a critical link between centrosome/ciliary biology and nuclear functions, particularly through its interactions with chromatin remodeling complexes.

What are the main applications for OFD1 antibody, HRP conjugated in experimental settings?

The HRP-conjugated OFD1 antibody is particularly useful for:

• Western blot analysis to detect endogenous and recombinant OFD1 protein

• Immunoprecipitation studies to investigate OFD1's protein-protein interactions

• Immunohistochemistry to examine OFD1 localization in tissue sections

• ELISA-based detection and quantification of OFD1 protein

The HRP conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibody incubation steps in many protocols. This antibody is especially valuable for co-immunoprecipitation experiments investigating OFD1's interactions with proteins like RuvBl1 and components of the TIP60 complex .

How should I validate the specificity of an OFD1 antibody before experimental use?

Methodological approach for antibody validation:

• Positive controls: Use cell lines known to express OFD1 (e.g., MDCK cells as used in the literature)

• Negative controls: Use OFD1 knockout/knockdown cells or tissues

• Peptide competition: Pre-incubate the antibody with the immunogenic peptide to confirm specificity, as demonstrated in the literature where preincubated anti-OFD1 was unable to immunoprecipitate the OFD1 protein

• Multiple detection methods: Confirm OFD1 detection using different techniques (e.g., immunofluorescence, Western blot)

• Molecular weight confirmation: Verify that the detected protein corresponds to the expected molecular weight of OFD1 (approximately 110 kDa, though multiple bands may be present due to alternative splicing)

What sample preparation protocols optimize OFD1 detection in Western blot applications?

For optimal OFD1 detection in Western blots:

• Cell lysis: Use a lysis buffer containing 1% Triton X-100, 150mM NaCl, 50mM Tris-HCl pH 7.5, and protease inhibitors

• Protein denaturation: Heat samples at 95°C for 5 minutes in Laemmli sample buffer with β-mercaptoethanol

• Gel selection: Use 8-10% polyacrylamide gels for optimal separation of OFD1 (approximately 110 kDa)

• Protein transfer: Transfer to PVDF membranes at 100V for 90 minutes in cold transfer buffer with 20% methanol

• Blocking: Block membranes with 5% non-fat dry milk in TBS-T for 1 hour at room temperature

• Antibody dilution: Dilute HRP-conjugated OFD1 antibody in 1% BSA in TBS-T

• Detection enhancement: For weak signals, consider using enhanced chemiluminescence substrates with extended exposure times

This protocol has been optimized based on successful detection of OFD1 in experimental systems described in the literature .

How should I design co-immunoprecipitation experiments to study OFD1 protein interactions?

Based on successful co-immunoprecipitation experiments in the literature , follow this methodological approach:

• Cell preparation: Use cells expressing both OFD1 and your protein of interest (either endogenous or tagged versions)

• Crosslinking consideration: Consider using reversible crosslinkers like dithiobis-succinimidyl propionate (DSP) to stabilize transient protein-protein interactions, as this approach enhanced detection of OFD1-Nro1 interactions

• Lysis conditions: Use gentle lysis buffers (e.g., 20mM HEPES pH 7.4, 150mM NaCl, 0.5% NP-40) with protease inhibitors

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Antibody binding: Incubate lysates with HRP-conjugated OFD1 antibody overnight at 4°C

• Elution strategies: For detection of co-immunoprecipitated proteins with similar molecular weights as IgG heavy chains (approximately 50 kDa), consider peptide elution as described in the literature to eliminate IgG contamination

• Controls: Include IgG controls and use peptide competition as negative controls

This approach has successfully identified interactions between OFD1 and proteins such as RuvBl1, RuvBl2, DMAP1, TRRAP, and TIP60 .

How can I investigate OFD1's dual localization to centrosomes and nucleus using the HRP-conjugated antibody?

To investigate the dual localization of OFD1:

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions using differential centrifugation

  • Further purify centrosomal fractions using sucrose gradient ultracentrifugation

  • Analyze fractions by Western blot using HRP-conjugated OFD1 antibody

  • Include markers for different cellular compartments (e.g., lamin for nucleus, γ-tubulin for centrosomes)

• Immunofluorescence with signal amplification:

  • Fix cells with 4% paraformaldehyde followed by permeabilization

  • Block with 5% BSA

  • Incubate with OFD1 antibody

  • For HRP-conjugated antibodies, use tyramide signal amplification to visualize

  • Co-stain with markers for centrosomes (γ-tubulin) and nucleus (DAPI)

  • Analyze by confocal microscopy to confirm the dual localization reported in the literature

• Proximity ligation assay:

  • To investigate OFD1's interactions with specific proteins in different cellular compartments

  • Use OFD1 antibody in conjunction with antibodies against known interactors like RuvBl1

  • This method will reveal where in the cell these interactions occur

What methods can I use to study the oxygen-dependent regulation of OFD1 function?

Based on research showing oxygen-dependent regulation of Ofd1 in fission yeast , consider these methodological approaches:

• Hypoxia chambers:

  • Culture cells in controlled oxygen environments (1-5% O₂)

  • Harvest at different time points for analysis of OFD1 interactions

  • Use HRP-conjugated OFD1 antibody for Western blot and co-IP experiments

• Prolyl hydroxylase domain inhibition:

  • Treat cells with prolyl hydroxylase inhibitors (e.g., DMOG or CoCl₂)

  • Compare OFD1 interactions under normoxic conditions with and without inhibitors

  • Analyze using co-IP with HRP-conjugated OFD1 antibody

• Mutational analysis:

  • Generate mutations in the OFD1 dioxygenase domain

  • Analyze effects on protein-protein interactions under different oxygen conditions

  • This approach is supported by findings that Ofd1 iron-coordinating mutants affect protein interactions

• Mass spectrometry:

  • Perform immunoprecipitation with HRP-conjugated OFD1 antibody

  • Compare interactomes under normoxic and hypoxic conditions

  • Identify oxygen-dependent changes in OFD1-containing complexes

What are the common pitfalls when detecting OFD1 protein and how can they be addressed?

How should I interpret differences in OFD1 detection patterns when studying its interaction with the TIP60 complex?

When analyzing OFD1's interaction with the TIP60 complex:

• Detection of multiple complex components: Successful experiments should detect co-immunoprecipitation of multiple TIP60 complex components (RuvBl1, RuvBl2, DMAP1, TRRAP, and TIP60) as shown in the literature

• Reciprocal co-IP validation: Confirm interactions by performing reciprocal co-IPs (e.g., IP with anti-TIP60 to detect OFD1, as demonstrated in research)

• Subcellular localization correlation: Correlate co-IP data with subcellular localization studies to confirm where these interactions occur (primarily nuclear)

• Interaction dynamics interpretation:

  • Strong OFD1-TIP60 complex interactions suggest activation of chromatin remodeling

  • Weak or absent interactions may indicate disruption of nuclear functions

  • Changes in interaction patterns following treatments may reveal regulatory mechanisms

• Data verification approaches:

  • Use peptide elution to eliminate IgG contamination

  • Include appropriate controls (peptide competition, IgG controls)

  • Verify results with multiple detection methods

How can I study the homotypic interaction domain of OFD1 using HRP-conjugated antibodies?

To investigate OFD1 self-association through its coiled-coil domains :

• Co-immunoprecipitation of differently tagged OFD1 variants:

  • Co-express differentially tagged OFD1 constructs (e.g., Flag-OFD1 and MycGFP-OFD1)

  • Perform co-IP using anti-tag antibodies

  • Detect using HRP-conjugated OFD1 antibody as an alternative detection method

  • This approach successfully demonstrated OFD1 self-association in previous research

• Domain mapping experiments:

  • Generate OFD1 truncation constructs focusing on the CC domains

  • Perform interaction studies to identify critical regions for self-association

  • Research has shown the fourth CC domain is critical for homotypic interactions

• Yeast two-hybrid system:

  • Use OFD1 fragments as both bait and prey

  • Map interaction domains through systematic deletion analysis

  • Validate findings in mammalian cells using co-IP with HRP-conjugated OFD1 antibody

• GST pulldown assays:

  • Express GST-tagged OFD1 domains

  • Test interaction with other OFD1 constructs

  • Detect using HRP-conjugated OFD1 antibody

What methodological approaches can reveal the functional significance of OFD1's interaction with RuvBl1?

Based on the identified interaction between OFD1 and RuvBl1 , consider these advanced methodological approaches:

• Functional domain analysis:

  • Map the interaction domains using truncated constructs of both proteins

  • Research has shown that OFD1's central CC region and C-terminal region are involved in RuvBl1 binding

  • Use site-directed mutagenesis to create specific point mutations

  • Analyze interaction using co-IP with HRP-conjugated OFD1 antibody

• Chromatin immunoprecipitation (ChIP) assays:

  • Investigate whether OFD1-RuvBl1 complexes associate with specific genomic regions

  • Compare binding patterns under normal and stress conditions

  • Relate findings to the known role of RuvBl1 in chromatin remodeling complexes

• Functional knockdown/knockout studies:

  • Deplete RuvBl1 and analyze effects on OFD1 localization and function

  • Deplete OFD1 and analyze effects on RuvBl1 activity and localization

  • Rescue experiments using wild-type and mutant constructs

  • Detect proteins using HRP-conjugated antibodies for direct visualization

• Proximity-dependent biotin identification (BioID):

  • Create fusion proteins of OFD1 or RuvBl1 with a biotin ligase

  • Identify proteins in close proximity in living cells

  • Compare interactome under different conditions

  • Validate key interactions using HRP-conjugated OFD1 antibody