NR2F1 Antibody, Biotin conjugated

What is NR2F1 and why is it important in cellular localization studies?

NR2F1 (Nuclear receptor subfamily 2, group F, member 1), also known as COUP-TF1, is an orphan nuclear receptor belonging to the superfamily of steroid/thyroid hormone receptors that functions as a strong transcriptional regulator. This protein contains a DNA binding domain (DBD) with two conserved zinc-finger motifs and primarily localizes to the nucleus where it regulates gene expression .

The significance of NR2F1 in cellular localization studies stems from its role in binding to the ovalbumin promoter and stimulating transcription initiation in conjunction with other proteins. It specifically binds to both direct repeats and palindromes of the 5'-AGGTCA-3' motif and represses transcriptional activity of LHCG . Recent research has shown that NR2F1 primarily localizes to the nucleoplasm, contrary to some reports suggesting nucleolar localization, which appears to be an antibody-specific artifact .

What are the most reliable methods for validating NR2F1 antibody specificity in research?

Validating NR2F1 antibody specificity requires a multi-faceted approach:

• CRISPR/Cas9 engineered null cells: Testing antibodies in both wild-type and CRISPR/Cas9-engineered cells lacking NR2F1 expression provides definitive evidence of specificity. This approach revealed that some apparent nucleolar staining patterns were artifacts .

• Multiple antibody comparison: Systematically testing multiple anti-NR2F1 antibodies (at least 5-7 different ones) using the same protocol across various applications. This approach identified which antibodies produced consistent nuclear staining versus those that produced artifactual nucleolar patterns .

• Recombinant protein expression: Using NR2F1-GFP fusion proteins to verify localization patterns independent of antibody staining. This method confirmed the predominant nucleoplasmic localization of NR2F1 without nucleolar staining .

• Bioinformatic validation: Analyzing publicly available datasets and algorithms to predict biophysical and functional domains of NR2F1, along with examining scRNA-seq data to determine co-expression patterns with potential interactors .

• Western blotting confirmation: Verifying antibody specificity by western blot, where properly validated NR2F1 antibodies should detect bands at the expected molecular weight (approximately 46 kDa) .

How can researchers distinguish between true NR2F1 localization and antibody artifacts?

Distinguishing true NR2F1 localization from artifacts requires careful experimental design:

A systematic study of seven commonly used anti-NR2F1 antibodies revealed that the mouse monoclonal antibody (clone H8132) produced nucleolar-like staining that was determined to be artifactual. This staining pattern appeared even in cells that do not express NR2F1, such as hiPSCs. Researchers should be particularly cautious when using this antibody for quantification studies .

Multiple evidence sources, including GFP-tagged NR2F1 expression, bioinformatic analysis of protein domains, and testing in NR2F1-null cells, confirm that genuine NR2F1 localization is diffusely distributed in the nucleoplasm without nucleolar aggregates .

What are the advantages and limitations of different biotinylation methods for antibodies?

Different biotinylation methods offer distinct advantages and limitations for research applications:

ZBPA Domain Method:

• Advantages: Provides stringent antibody biotinylation by specifically targeting the Fc region, prevents unwanted protein biotinylation, produces more specific immunostaining with less background .

• Limitations: Generally requires higher antibody concentrations to achieve similar staining intensity as other methods, may result in some antibody loss during filtering steps .

Lightning-Link Method:

• Advantages: Rapid conjugation protocol, works with lower antibody concentrations .

• Limitations: Can result in biotinylation of stabilizer proteins (e.g., HSA, gelatin) leading to non-specific nuclear and cytoplasmic staining in many tissues, creating potential background issues .

LYNX Rapid Plus Biotin Conjugation:

• Advantages: Enables rapid conjugation in minutes, allows conjugation at near-neutral pH with 100% antibody recovery, no requirement for desalting or dialysis .

• Limitations: Type-specific (Type 1 vs. Type 2) kits must be carefully selected based on whether the conjugate will be captured by streptavidin-labeled plates or complexed with streptavidin detection reagents .

Abcam Biotin Fast Conjugation Kit:

• Advantages: One-step procedure with only 30 seconds hands-on time, conjugates ready in less than 20 minutes, optimized for use with streptavidin-labeled detection reagents .

• Limitations: Specific kit sizes must be selected based on the amount of antibody to be labeled .

What controls should be included when using NR2F1 Antibody, Biotin conjugated in immunohistochemistry?

For rigorous immunohistochemistry experiments with NR2F1 Antibody, Biotin conjugated, researchers should include the following controls:

• Primary antibody only control: Staining with only the NR2F1 primary antibody to establish the baseline staining pattern without biotin conjugation .

• Secondary antibody only control: Using only secondary antibody without primary to identify potential non-specific binding of the detection system .

• Isotype control: Using an irrelevant antibody of the same isotype and host species (e.g., rabbit IgG for rabbit polyclonal NR2F1 antibodies) to identify non-specific binding .

• Biotinylated buffer protein control: Testing biotinylated stabilizer proteins (HSA, gelatin) using the same detection method to identify background from non-antibody components .

• Free biotin removal control: Filtering antibodies to remove free biotin and comparing staining patterns before and after filtration .

• Knockout/knockdown tissue control: Using tissues from NR2F1 knockout or knockdown models as negative controls to confirm antibody specificity .

• Concentration titration: Testing multiple antibody dilutions to establish optimal signal-to-noise ratio .

• Multiple antibody comparison: Using alternative NR2F1 antibodies to verify staining patterns across different clones and manufacturers .

How does NR2F1 antibody performance compare across different applications?

NR2F1 antibody performance varies significantly across different experimental applications:

For Western blot applications, ZBPA-biotinylated antibodies performed similarly to their unconjugated counterparts, detecting proteins at expected molecular weights without altering their usability . For immunohistochemical and immunofluorescence applications, significant variability was observed between antibody clones, with some producing artifactual nucleolar staining patterns .

What are the best protocols for fixation when using NR2F1 Antibody, Biotin conjugated?

Fixation protocols significantly impact NR2F1 antibody staining patterns and can affect the detection of true versus artifactual signals:

Standard Paraformaldehyde (PFA) Fixation:

• Fix cells with 4% paraformaldehyde in PBS at 4°C overnight

• Wash 3× in wash buffer (0.1% Triton X-100 in PBS)

• Block in blocking buffer (PBS with 1% BSA and 0.1% Triton X-100)

• Incubate with primary NR2F1 antibody diluted in blocking buffer at 4°C overnight

• Wash with wash buffer

• Incubate with appropriate secondary detection system at room temperature for 1 hour

• Mount with 40% glycerol in PBS containing 2% 1,4-Diazabicyclo[2.2.2]

Alternative Fixation Protocol for Reducing Nucleolar Artifacts:

When using potentially artifact-prone antibodies (e.g., clone H8132), researchers should test multiple fixation protocols as the nucleolar-like staining patterns may be dependent on fixation methods . A systematic comparison of multiple antibody concentrations (e.g., 2 µg/ml, 1 µg/ml, and 0.4 µg/ml) is recommended to determine optimal conditions for specific versus non-specific staining .

It's important to note that fixation with formaldehyde (FA) can weaken GFP signals when using NR2F1-GFP fusion proteins as controls, potentially requiring anti-GFP antibody enhancement .

How can NR2F1 Antibody, Biotin conjugated be optimized for multiplex immunofluorescence studies?

Optimizing NR2F1 Antibody, Biotin conjugated for multiplex immunofluorescence requires careful consideration of several factors:

• Antibody selection: Choose NR2F1 antibodies validated for specificity in immunofluorescence applications. Avoid clone H8132 which produces nucleolar artifacts unless specifically studying this pattern .

• Biotinylation method: Consider using the ZBPA biotinylation method which provides more specific staining compared to Lightning-Link, especially when multiplex studies require minimal background .

• Detection strategy: For multiple antibodies from the same species, use distinct conjugate molecules that can be targeted by different secondary antibodies. The ZBPA technique offers the possibility of conjugating various molecules other than biotin to an antibody .

• Sequential staining approach: When using multiple primary antibodies in the same species, employ sequential staining with intermediate blocking steps to prevent cross-reactivity .

• Co-staining validation: Validate co-staining patterns by comparing with single-stained controls to ensure antibody combinations don't interfere with each other's binding or create artifacts .

• Concentration optimization: Titrate the concentration of NR2F1 Antibody, Biotin conjugated to achieve optimal signal-to-noise ratio in the multiplex context, as the presence of other antibodies may affect binding efficiency .

• Signal amplification adjustments: When using streptavidin-based detection systems, adjust the concentration of streptavidin conjugates to balance signal intensity across all fluorescence channels .

What are the molecular mechanisms behind NR2F1 function that impact antibody selection?

Understanding NR2F1's molecular structure and function is crucial for proper antibody selection:

NR2F1 functions as a transcription factor that binds to thousands of targets including genes and their enhancers . It contains distinct functional domains that should be considered when selecting antibodies:

• DNA Binding Domain (DBD): Contains two conserved zinc-finger motifs responsible for recognizing specific DNA sequences. Antibodies targeting this region may interfere with DNA binding in chromatin immunoprecipitation (ChIP) experiments .

• N-terminal Disordered Domain: NR2F1 contains a highly disordered domain at the N-terminus. Antibodies targeting disordered regions may show different binding characteristics depending on protein conformation .

• Protein Interaction Sites: NR2F1 interacts with various proteins including TFAP2A. Antibodies targeting interaction surfaces may block protein-protein interactions or show reduced binding when NR2F1 is engaged in complexes .

• Nucleoplasmic Localization: Genuine NR2F1 primarily localizes to the nucleoplasm, not nucleoli. Antibodies showing nucleolar patterns (like clone H8132) are likely detecting artifacts rather than true localization .

The choice of antibody should be guided by:

• The specific region of NR2F1 used as immunogen (N-terminal vs. C-terminal vs. internal regions)

• Whether the target epitope is accessible in the experimental context

• Whether the antibody has been validated in the specific application (WB, IHC, IF, ChIP)

• The compatibility of the epitope with biotinylation without affecting antibody function

How does long non-coding RNA NR2F1-AS1 research impact the use of NR2F1 antibodies?

Recent research on the long non-coding RNA NR2F1-AS1 (NAS1) presents important considerations for NR2F1 antibody applications:

NR2F1-AS1 has been identified as upregulated in dormant mesenchymal-like breast cancer stem cells and functionally promotes tumor dissemination . This research has several implications for NR2F1 antibody applications:

• Regulatory relationship: NR2F1-AS1 knockdown results in transcriptomic changes toward a different cellular state, suggesting a potential regulatory relationship with NR2F1 expression or function . Researchers studying NR2F1 should consider measuring NR2F1-AS1 levels to account for potential regulatory effects.

• Expression correlation: When using NR2F1 antibodies in cancer research, particularly in metastasis studies, researchers should validate whether observed patterns correlate with NR2F1-AS1 expression which may influence interpretation of results .

• Functional validation: Beyond simple detection of NR2F1 protein levels with antibodies, functional validation through knockdown experiments similar to those performed with NR2F1-AS1 may be necessary to establish causality in observed phenotypes .

• Metastatic dormancy models: When using NR2F1 antibodies in metastatic dormancy research, researchers should consider the influence of NR2F1-AS1 on cellular behavior, as knockdown studies showed fewer GFP+ cancer cells initially seeded in the lungs after NR2F1-AS1 knockdown .

• Potential therapeutic targets: The relationship between NR2F1 and its antisense transcript suggests complex regulatory mechanisms that could impact therapeutic approaches targeting either molecule, necessitating careful antibody-based validation of both factors in experimental models .