DOF3.4 Antibody

What is DOF3.4/OBP1 protein and what are its functions in plant biology?

DOF3.4 (also known as OBP1 or OBF binding protein 1) is a Dof zinc finger protein transcription factor found in Arabidopsis thaliana (Mouse-ear cress). This protein belongs to the Dof (DNA binding with One Finger) family of plant-specific transcription factors that regulate various physiological processes in plants.

DOF3.4/OBP1 functions include:

• Regulation of gene expression through DNA binding

• Involvement in plant development pathways

• Potential role in stress responses

The protein is identified by UniProt accession number Q39088 and is encoded by the gene AT3G50410 . Understanding its function is crucial for plant molecular biology research focusing on transcriptional regulation mechanisms.

What validation methods should be employed to confirm DOF3.4 Antibody specificity?

Antibody validation is critical for ensuring experimental rigor. For DOF3.4 Antibody, researchers should implement multiple validation strategies following the five pillars approach recommended by the International Working Group for Antibody Validation:

• Orthogonal method validation: Compare antibody results with mass spectrometry or RNA-seq data to verify DOF3.4 protein expression patterns.

• Genetic validation: Test the antibody in DOF3.4 knockdown or knockout Arabidopsis lines. The signal should be significantly reduced or absent in these samples compared to wild-type plants.

• Independent antibody validation: Use at least two different DOF3.4 antibodies targeting distinct epitopes and compare their recognition patterns.

• Expression validation: Test the antibody against recombinant DOF3.4 protein expressed in a heterologous system.

• Immunoprecipitation-mass spectrometry: Confirm that the antibody pulls down the correct protein through mass spectrometry analysis .

These validation methods are essential as demonstrated by YCharOS findings, which showed that >50% of commercial antibodies failed characterization experiments in at least one common application .

What are the recommended technical protocols for using DOF3.4 Antibody in Western blots?

For optimal Western blot results with DOF3.4 Antibody:

Sample Preparation:

• Extract proteins from plant tissues using a buffer containing phosphatase and protease inhibitors

• For nuclear proteins like DOF3.4, use nuclear extraction protocols

• Load 20-40 μg of total protein per lane

Western Blot Protocol:

• Separate proteins on 10-12% SDS-PAGE gel

• Transfer to PVDF or nitrocellulose membrane (PVDF preferred for transcription factors)

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

• Incubate with DOF3.4 Antibody at 1:1000 dilution overnight at 4°C

• Wash 3× with TBST, 10 minutes each

• Incubate with secondary antibody (typically HRP-conjugated anti-rabbit IgG) at 1:5000 dilution

• Wash 3× with TBST, 10 minutes each

• Develop using ECL detection reagent

Controls:

• Positive control: Arabidopsis wild-type seedling nuclear extract

• Negative control: Extract from DOF3.4 knockout line

• Loading control: Anti-histone H3 antibody or other nuclear marker

How can DOF3.4 Antibody be used to study protein-protein interactions involving the DOF3.4 transcription factor?

To investigate protein-protein interactions involving DOF3.4:

Co-immunoprecipitation (Co-IP):

• Prepare plant nuclear extracts under non-denaturing conditions

• Pre-clear with Protein A/G beads

• Incubate cleared lysate with DOF3.4 Antibody (4-5 μg per 500 μg protein)

• Add Protein A/G beads and incubate overnight at 4°C

• Wash extensively to remove non-specific interactions

• Elute bound proteins and analyze by Western blot or mass spectrometry

Proximity Ligation Assay (PLA):
This technique allows visualization of protein-protein interactions in situ:

• Fix and permeabilize plant tissues

• Incubate with DOF3.4 Antibody and antibody against potential interacting partner

• Add PLA probes with complementary oligonucleotides

• Perform ligation and amplification

• Detect signals using fluorescence microscopy

Chromatin Immunoprecipitation (ChIP):
For studying DNA-protein interactions:

• Cross-link proteins to DNA in plant tissues

• Isolate and sonicate chromatin

• Immunoprecipitate with DOF3.4 Antibody

• Reverse cross-links and purify DNA

• Analyze by qPCR or sequencing to identify DOF3.4 binding sites

What are the critical differences between polyclonal and monoclonal DOF3.4 Antibodies for research applications?

Polyclonal DOF3.4 Antibodies:

• Recognize multiple epitopes on the DOF3.4 protein

• Generally provide higher sensitivity for detection

• Exhibit greater batch-to-batch variation

• Optimal for applications like immunoprecipitation and ChIP

• Often rabbit-derived (e.g., CSB-PA655098XA01DOA)

Monoclonal DOF3.4 Antibodies:

• Recognize a single epitope with high specificity

• Provide consistent results across experiments

• Show minimal batch-to-batch variation

• Preferred for quantitative applications

• May have lower sensitivity than polyclonals

Selection Criteria Table:

The choice depends on your specific experimental requirements and the expected expression level of DOF3.4 in your samples .

How should researchers troubleshoot non-specific binding or high background when using DOF3.4 Antibody?

When encountering high background or non-specific binding:

Systematic Troubleshooting Approach:

• Antibody Dilution Optimization:

  • Test a dilution series (1:500, 1:1000, 1:2000, 1:5000)

  • Higher dilutions may reduce non-specific binding

• Blocking Optimization:

  • Try different blocking agents (BSA, non-fat milk, commercial blockers)

  • Increase blocking time (2-3 hours instead of 1 hour)

• Buffer Modifications:

  • Increase salt concentration in wash buffers (up to 500 mM NaCl)

  • Add 0.1-0.5% Triton X-100 or Tween-20 to reduce hydrophobic interactions

  • Add 5% glycerol to reduce non-specific interactions

• Pre-adsorption Protocol:
  For high background in immunohistochemistry:

  1. Dilute antibody in buffer containing 1-5% of the blocking agent

  2. Add protein extract from non-target tissue

  3. Incubate at 4°C for 24 hours

  4. Centrifuge and use the supernatant

• Negative Controls:

  • Include samples from DOF3.4 knockout plants

  • Use pre-immune serum or isotype control antibodies

  • Omit primary antibody in parallel samples

How does the host species affect DOF3.4 Antibody performance in different experimental contexts?

The host species in which the DOF3.4 Antibody was raised significantly impacts its performance:

Rabbit-derived DOF3.4 Antibodies:

• Most commonly available for plant proteins (e.g., CSB-PA655098XA01DOA)

• Generally produce high-affinity antibodies with good sensitivity

• Preferred for Western blot and immunoprecipitation

• May show cross-reactivity with some plant proteins

• Higher background in certain plant tissues due to endogenous immunoglobulins

Mouse-derived DOF3.4 Antibodies:

• Less common for plant research antibodies

• Often produce more specific monoclonal antibodies

• Better for co-localization studies (can be paired with rabbit antibodies)

• May have lower sensitivity than rabbit polyclonals

• Good for flow cytometry and immunohistochemistry

Other Host Considerations:

• Goat or chicken antibodies can reduce background in certain plant tissues

• Camelid single-domain antibodies offer advantages for recognizing specific conformational epitopes

• The host should be selected based on the specific application and tissue being studied

What approaches can enhance detection sensitivity for low-abundance DOF3.4 protein?

For detecting low levels of DOF3.4 transcription factor:

Signal Amplification Methods:

• Tyramide Signal Amplification (TSA):

  • Can increase sensitivity by 10-100 fold

  • Use HRP-conjugated secondary antibody

  • Add tyramide substrate that deposits at the site of antibody binding

  • Particularly useful for immunohistochemistry

• Polymer-based Detection Systems:

  • Use secondary antibodies conjugated to dextran polymers carrying multiple HRP molecules

  • Provides signal enhancement without increasing background

• Biotin-Streptavidin Amplification:

  • Use biotinylated secondary antibody

  • Add streptavidin-conjugated fluorophore or enzyme

  • Provides 3-4 fold signal enhancement

Sample Enrichment Techniques:

• Nuclear Extraction:

  • Concentrate nuclear proteins to enrich for transcription factors

  • Use specific nuclear extraction buffers with protease inhibitors

• Immunoprecipitation Before Western Blot:

  • Concentrate DOF3.4 protein by immunoprecipitation

  • Analyze by Western blot for increased sensitivity

• Targeted Mass Spectrometry:

  • Use immunocapture coupled with targeted mass spectrometry

  • Can provide femtomole-level sensitivity

How can DOF3.4 Antibody be used to study post-translational modifications of the DOF3.4 protein?

To investigate post-translational modifications (PTMs) of DOF3.4:

Phosphorylation Analysis:

• Use phospho-specific DOF3.4 antibodies (if available)

• Alternatively, immunoprecipitate with general DOF3.4 Antibody then probe with anti-phosphoserine/threonine/tyrosine antibodies

• Treat samples with phosphatase before Western blot to confirm phosphorylation

• Use Phos-tag™ SDS-PAGE to separate phosphorylated forms

Ubiquitination Analysis:

• Immunoprecipitate with DOF3.4 Antibody

• Probe with anti-ubiquitin antibody

• Use proteasome inhibitors to stabilize ubiquitinated forms

• Compare molecular weight shifts to identify ubiquitination

Mass Spectrometry Approach:

• Immunoprecipitate DOF3.4 using the antibody

• Perform tryptic digestion

• Analyze by LC-MS/MS with PTM-specific fragmentation methods

• Compare modified and unmodified peptides

• Quantify PTM abundance across different conditions

The methodology is similar to that used for RAS network proteins, where antibodies enabled detection of 27 phosphopeptides from various target proteins .

What considerations are important for cross-species reactivity of DOF3.4 Antibody?

When considering DOF3.4 Antibody use across different plant species:

Epitope Conservation Analysis:

• Perform sequence alignment of DOF3.4 homologs across target species

• Identify the epitope recognized by the antibody

• Assess sequence conservation in this region

• Higher conservation suggests better cross-reactivity

Validation Requirements for Cross-Species Use:

• Always test the antibody in the new species before extensive experiments

• Include positive control from Arabidopsis thaliana

• Compare band patterns and molecular weights

• Verify specificity using genetic approaches if possible

Cross-Reactivity Testing Protocol:

• Run Western blots with protein extracts from multiple species

• Include concentration gradients to assess sensitivity

• Perform immunoprecipitation followed by mass spectrometry to confirm target identity

• Test preabsorption with recombinant DOF3.4 protein to confirm specificity

Predicted Cross-Reactivity Table: