NUBP2 Antibody, Biotin conjugated

What is NUBP2 and what is its biological significance?

NUBP2 (Nucleotide Binding Protein 2) is a member of the NUBP/MRP gene subfamily of ATP-binding proteins. The protein contains conserved ATP/GTP binding motif A (P-loop), ATP/GTP binding motif A-prime, and NUBP/MRP alpha and beta motifs . Human NUBP2 is a 271-amino acid protein that shares 72.6% amino acid similarity with mouse Nubp2. Notably, NUBP2 lacks the additional N-terminal sequence with 4 cysteine residues that is present in NUBP1 .

Northern blot analysis has detected a 1.4-kb NUBP2 transcript with ubiquitous expression in all human adult and fetal tissues tested, with highest expression observed in adult skeletal muscle . This widespread expression pattern suggests NUBP2 plays essential roles in fundamental cellular processes. The protein functions as a cytosolic Fe-S cluster assembly factor, which is critical for various cellular activities including electron transport, metabolic reactions, and sensing for regulatory processes.

What are the available applications for NUBP2 Antibody (Biotin conjugated)?

NUBP2 Antibody (Biotin conjugated) has been validated for multiple experimental applications, providing researchers with versatility in their studies:

• Western Blotting (WB): For protein detection and quantification in cell/tissue lysates

• ELISA: For quantitative analysis of NUBP2 expression

• Immunohistochemistry (Frozen Sections) (IHC-F): For localization studies in frozen tissue sections

• Immunohistochemistry (Paraffin-embedded Sections) (IHC-P): For detection in fixed paraffin-embedded tissues

The antibody has been purified using Protein A purification methods, ensuring high purity for these applications . The biotin conjugation provides significant flexibility, allowing detection via streptavidin-based systems which can enhance sensitivity in various experimental contexts.

How does the host species and clonality affect experimental design?

The NUBP2 Antibody (Biotin conjugated) is a rabbit-hosted polyclonal antibody . This has several methodological implications for researchers:

• Polyclonal nature: The antibody recognizes multiple epitopes within the amino acid region 25-110 of human NUBP2 . This provides advantages in signal amplification and robustness across different sample preparation conditions, but researchers should account for potential batch-to-batch variation.

• Host considerations: The rabbit origin must be considered when designing multi-antibody experiments to avoid cross-reactivity. Secondary detection systems must be compatible with rabbit-originated primary antibodies.

• Experimental controls: When designing experiments, include appropriate negative controls (non-immune rabbit IgG) and positive controls (tissues known to express NUBP2) to validate results.

• Cross-species applications: While targeted against human NUBP2, the antibody has predicted reactivity with mouse, rat, cow, sheep, pig, and horse samples . Cross-reactivity validation is recommended before extensive use with non-human samples.

What are the storage and handling recommendations for maintaining antibody activity?

For optimal antibody performance and stability:

• Upon receipt, store at -20°C or -80°C to maintain long-term activity .

• Avoid repeated freeze-thaw cycles which can degrade antibody performance. Aliquot upon receipt if multiple uses are planned.

• For short-term use (up to one month), storage at 4°C is acceptable.

• The antibody is typically provided in buffer containing glycerol (often 50%) and preservatives like Proclin 300 (0.03%) , maintaining stability during proper storage.

• Prior to use, allow the antibody to equilibrate to room temperature and gently mix - avoid vigorous shaking or vortexing which can denature antibodies.

• Working dilutions should be prepared fresh for optimal results.

What optimization strategies are recommended for Western blotting with NUBP2 Antibody?

Western blotting with biotin-conjugated NUBP2 antibody requires careful optimization:

• Sample preparation:

  • Complete protein denaturation is essential - include reducing agents in sample buffer

  • For cell/tissue lysates, include protease inhibitors to prevent NUBP2 degradation

  • Optimal protein loading concentration should be determined empirically (typically start with 20-40 μg)

• Transfer optimization:

  • Given that NUBP2 is approximately 31 kDa, semi-dry transfer systems with PVDF membranes typically work well

  • Transfer time of 60-90 minutes at 15-20V is a good starting point

• Detection strategy:

  • Since the antibody is biotin-conjugated, use streptavidin-HRP for detection

  • Titrate the antibody concentration (recommended starting dilution: 1:1000 to 1:2000)

  • Include longer blocking steps (1-2 hours) to reduce background from biotin-based detection

  • Consider using casein-based blockers instead of milk to reduce endogenous biotin interference

• Control recommendations:

  • Include recombinant NUBP2 as positive control

  • Run samples from tissues known to have high NUBP2 expression (e.g., skeletal muscle) as biological controls

What are effective validation methods to confirm NUBP2 Antibody specificity?

Antibody validation is critical for ensuring research reproducibility. For NUBP2 antibody, consider:

• Peptide blocking experiments:

  • Pre-incubate the antibody with the immunizing peptide (derived from human NUBP2 AA 25-110)

  • Reduction or elimination of signal confirms specificity

• Genetic validation:

  • Compare signal between wild-type samples and NUBP2 knockdown/knockout models

  • Use CRISPR-Cas9 to generate NUBP2-null cell lines for negative controls

• Orthogonal validation:

  • Compare protein detection results with mRNA expression (qPCR)

  • Use multiple antibodies targeting different NUBP2 epitopes

• Cross-platform validation:

  • Confirm localization pattern across multiple techniques (IHC, IF, WB)

  • Verify expected molecular weight in Western blots (approximately 31 kDa)

The strategies above align with recent guidelines for antibody validation described in literature, where multiple lines of evidence are recommended to establish specificity .

How can researchers effectively troubleshoot cross-reactivity issues?

Cross-reactivity troubleshooting requires systematic investigation:

• Identifying potential cross-reactants:

  • Sequence homology analysis between NUBP2 and related proteins like NUBP1

  • In silico epitope mapping to identify potential shared epitopes

• Experimental optimization:

  • Increase antibody dilution to reduce non-specific binding

  • Optimize blocking steps (longer duration, different blocking agents)

  • Include detergents in washing steps at appropriate concentrations (0.1-0.3% Tween-20)

• Sample-specific considerations:

  • For tissues with high endogenous biotin (liver, kidney), implement avidin/biotin blocking systems

  • Use species-appropriate blocking reagents (when investigating cross-species reactivity)

• Advanced strategies:

  • Implement peptide competition assays with peptides from potential cross-reactive proteins

  • Conduct comparative analysis with tissues/cells known to express or lack NUBP2

When interpreting cross-reactivity patterns, consider that this antibody's specificity spans amino acids 25-110 of human NUBP2 , which might share homology with other proteins containing similar domains.

What considerations are important for quantitative ELISA using NUBP2 Antibody?

For quantitative ELISA applications:

• Assay design considerations:

  • For sandwich ELISA, pair with a non-biotin conjugated NUBP2 antibody targeting a different epitope

  • For direct ELISA, coating concentration and buffers must be optimized for NUBP2 antigen

• Optimization protocol:

  • Generate standard curves using recombinant NUBP2 protein (concentration range: 0-500 ng/mL)

  • Determine optimal antibody dilution through checkerboard titration

  • Test multiple blocking agents to identify lowest background (BSA, casein, commercial blockers)

• Sample preparation:

  • Standardize protein extraction methods across experimental samples

  • Include protease inhibitors in lysis buffers

  • Consider sample dilution series to ensure readings fall within the linear range

• Signal development:

  • Since the antibody is biotin-conjugated, use streptavidin-HRP systems for detection

  • TMB substrates offer good sensitivity and dynamic range

A typical sandwich ELISA protocol would involve pre-coating wells with a capture antibody specific for NUBP2, adding samples, then detecting with the biotin-conjugated NUBP2 antibody, followed by streptavidin-HRP and substrate addition .

What are the optimal immunohistochemistry protocols for different tissue types?

Tissue-specific IHC optimization for NUBP2 detection:

• Tissue fixation and processing:

  • For formalin-fixed paraffin-embedded (FFPE) tissues: Optimal fixation time is 24-48 hours

  • For frozen sections: Rapid freezing in OCT compound followed by 10 μm sectioning

• Antigen retrieval methods:

  • FFPE sections typically require heat-induced epitope retrieval

  • Recommended starting conditions: 10 mM citrate buffer (pH 6.0), 95°C for 20 minutes

  • Alternative: Tris-EDTA buffer (pH 9.0) if citrate buffer yields insufficient results

• Tissue-specific considerations:

  • For skeletal muscle (high NUBP2 expression): Reduce antibody concentration to 1:200-1:400

  • For tissues with high endogenous biotin: Implement avidin/biotin blocking steps

  • For tissues with high background: Extended blocking (2+ hours) with 5-10% normal serum

• Detection protocol variations:

  • For low-expressing tissues: Implement tyramide signal amplification systems

  • For co-localization studies: Optimize sequential staining protocols with other antibodies

The antibody has been validated for both frozen and paraffin-embedded section immunohistochemistry , providing flexibility for different experimental needs.

How should researchers integrate NUBP2 Antibody into multi-color immunofluorescence studies?

Multi-color immunofluorescence requires careful experimental design:

• Fluorophore selection considerations:

  • Since the antibody is biotin-conjugated, streptavidin-fluorophore conjugates provide flexibility

  • Recommended fluorophores for multi-color experiments:

    • Streptavidin-Alexa Fluor 488 (green channel)

    • Streptavidin-Alexa Fluor 555/568 (red channel)

    • Streptavidin-Alexa Fluor 647 (far-red channel)

• Multiplexing protocols:

  • Sequential staining recommended when using multiple primary antibodies

  • Complete first antibody staining through streptavidin-fluorophore step

  • Block remaining biotin sites with excess unconjugated streptavidin

  • Proceed with next antibody

• Controls for multi-color experiments:

  • Single-stain controls to assess bleed-through

  • Fluorescence minus one (FMO) controls

  • Secondary-only controls to assess non-specific binding

• Advanced methods:

  • Spectral unmixing for closely overlapping fluorophores

  • Sequential scanning for confocal microscopy

This approach allows for effective colocalization studies of NUBP2 with other proteins of interest, providing insights into potential functional relationships.

What strategies can resolve contradictory data when comparing NUBP2 detection methods?

When facing contradictory results across detection methods:

• Systematic validation approach:

  • Compare protein abundance detected by Western blot vs. ELISA vs. IHC

  • Evaluate mRNA expression (qPCR) as an orthogonal measurement

  • Implement multiple antibodies targeting different NUBP2 epitopes

• Technical considerations:

  • Different sensitivities: ELISA typically more sensitive than Western blot

  • Different epitope accessibility: Native structure (ELISA) vs. denatured (Western blot)

  • Different quantification methods: Relative (Western) vs. absolute (ELISA)

• Analytical resolution framework:

  • Establish hierarchical decision tree based on assay robustness

  • Design targeted experiments to address specific contradictions

  • Consider biological context (tissue type, experimental conditions)

• Method-specific troubleshooting:

  • For Western blot: Optimize lysis conditions, reducing agents, transfer efficiency

  • For ELISA: Test different coating strategies, blocking agents, detection systems

  • For IHC: Compare different fixation methods, antigen retrieval, detection systems

Understanding method-specific limitations is crucial for appropriate data interpretation, particularly when working with complex target proteins like NUBP2 that may exist in different isoforms or post-translational modifications states.

How can researchers design robust experiments to study NUBP2 interactions with Fe-S cluster assembly?

Given NUBP2's role in Fe-S cluster assembly:

• Experimental design strategies:

  • Co-immunoprecipitation using biotin-conjugated NUBP2 antibody with streptavidin beads

  • Proximity ligation assays to detect NUBP2 interactions with other Fe-S assembly proteins

  • CRISPR-based knockout followed by rescue experiments with mutant NUBP2 variants

• Functional assay recommendations:

  • Enzymatic activity measurements of Fe-S dependent enzymes (aconitase, SDH)

  • Iron incorporation assays using radiolabeled iron

  • Cellular iron homeostasis measurements

• Technical approach:

  Technique	Application	Controls	Expected Outcome
  Co-IP with NUBP2 antibody	Identify interacting partners	IgG control, NUBP2 knockout	Detection of known Fe-S assembly proteins
  Western blot of fractionated samples	Determine subcellular localization	Fraction markers	Predominantly cytosolic signal
  ELISA after iron depletion/repletion	Quantify expression changes	Untreated cells	Potential upregulation during iron stress
  IHC of tissues with mitochondrial dysfunction	Assess response to stress	Normal tissues	Altered expression/localization pattern

• Data interpretation framework:

  • Compare results across multiple cell types/tissues

  • Evaluate context dependency of interactions

  • Correlate findings with established Fe-S assembly pathway models

This approach provides a comprehensive framework for investigating NUBP2's role in the complex process of Fe-S cluster assembly and its potential interactions with other proteins in this pathway.

What are emerging research applications for NUBP2 Antibody in studying cellular functions?

Recent developments suggest several promising research directions:

• Iron metabolism disorders:

  • NUBP2 detection in patient samples with conditions like Friedrich's ataxia

  • Correlation of NUBP2 expression with biomarkers of iron overload/deficiency

  • Potential diagnostic applications in mitochondrial disorders

• Cancer research applications:

  • Expression analysis across tumor types and correlation with prognosis

  • Investigation of NUBP2 in metabolic reprogramming of cancer cells

  • Potential therapeutic targeting of Fe-S assembly pathways

• Developmental biology:

  • Temporal expression patterns during embryonic development

  • Tissue-specific expression during differentiation

  • Role in stem cell maintenance and differentiation

• Stress response mechanisms:

  • NUBP2 modulation during oxidative stress

  • Response to hypoxia and relationship to HIF pathways

  • Involvement in cellular adaptation to nutrient limitation

The biotin-conjugated format of this antibody facilitates many of these applications through its compatibility with sensitive detection systems and potential for use in high-throughput screening approaches .

How can researchers evaluate the long-term reproducibility of experiments using NUBP2 Antibody?

Ensuring long-term reproducibility requires systematic approach:

• Documentation recommendations:

  • Maintain detailed records of antibody lot numbers, storage conditions

  • Document complete protocols including all buffers and incubation times

  • Implement electronic laboratory notebooks with standardized templates

• Quality control measures:

  • Establish internal reference standards for quantitative assays

  • Periodically validate antibody performance against known controls

  • Implement batch testing when receiving new antibody lots

• Statistical considerations:

  • Calculate intra- and inter-assay coefficients of variation

  • Establish acceptance criteria for experimental validation

  • Implement appropriate power calculations for sample size determination

• Reproducibility enhancement strategies:

  • Consider automated liquid handling for critical steps

  • Standardize image acquisition and analysis parameters

  • Implement blinding procedures for subjective assessments