NIFU5 Antibody

What is the NIFU5 Antibody and what organism does it target?

NIFU5 Antibody (Product Code: CSB-PA871687XA01DOA) is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana NIFU5 protein. It specifically targets the NIFU5 protein (UniProt No. Q9C8J2) in Arabidopsis thaliana (Mouse-ear cress), which is a model plant organism widely used in molecular biology research .

What are the optimal storage conditions for NIFU5 Antibody?

The antibody should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided as they can compromise antibody integrity and functionality. The antibody is supplied in liquid form in a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage .

How should freeze-thaw cycles be managed for NIFU5 Antibody?

To minimize the detrimental effects of freeze-thaw cycles, it's recommended to aliquot the antibody upon receipt into small volumes suitable for single-use experiments. Based on stability studies of similar antibodies, after thawing an aliquot, it should be kept at 4°C for short-term use. Extended exposure to room temperature or 37°C can significantly impact antibody activity, as demonstrated in stability studies for other research antibodies .

What validated applications exist for NIFU5 Antibody?

The NIFU5 Antibody has been tested and validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications. These methods allow for the detection and quantification of NIFU5 protein in plant samples . When designing experiments, researchers should consider the optimal antibody concentration for each application, which may require titration experiments.

What controls should be included when using NIFU5 Antibody in research?

Proper experimental design with NIFU5 Antibody should include:

• Positive control: Recombinant NIFU5 protein

• Negative control: Pre-immune serum

• Loading control: For Western blots, a housekeeping protein should be used

• Isotype control: A non-specific rabbit IgG at the same concentration

These controls help validate specificity and rule out non-specific binding, particularly important when working with polyclonal antibodies .

What is the recommended protocol for using NIFU5 Antibody in Western blotting?

While specific optimization may be required for NIFU5 Antibody, a general Western blot protocol would include:

• Sample preparation: Extract proteins from Arabidopsis thaliana tissues using an appropriate lysis buffer

• Protein separation: Run 10-30 μg protein on SDS-PAGE (10-12%)

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

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

• Primary antibody: Incubate with NIFU5 Antibody (1:500-1:2000 dilution) overnight at 4°C

• Washing: Wash 3-5 times with TBST

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit IgG (1:5000-1:10000) for 1 hour at room temperature

• Detection: Use enhanced chemiluminescence detection system

Optimization of antibody dilution and incubation conditions is recommended for each new lot of antibody .

How can NIFU5 Antibody be utilized in co-immunoprecipitation studies?

For co-immunoprecipitation (Co-IP) experiments with NIFU5 Antibody:

• Prepare plant cell lysate in a non-denaturing lysis buffer containing protease inhibitors

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

• Incubate 1-5 μg of NIFU5 Antibody with fresh lysate overnight at 4°C

• Add Protein A/G beads and incubate for 2-4 hours at 4°C

• Wash beads extensively (4-6 times) with IP wash buffer

• Elute bound proteins with SDS sample buffer

• Analyze by Western blot using antibodies against predicted interaction partners

This approach can help identify proteins that interact with NIFU5 in various plant physiological conditions or developmental stages.

How can surface plasmon resonance be used to analyze NIFU5 antibody-antigen binding kinetics?

Surface plasmon resonance (SPR) analysis can be used to determine binding kinetics between NIFU5 Antibody and its target:

• Immobilize purified NIFU5 protein on an AR2G sensor chip at 10 μg/ml in 10 mM sodium acetate (pH 4-6)

• Create a concentration series of NIFU5 Antibody (starting at ~50 nM with 1:2 dilutions)

• Perform binding analysis at 30°C with 1000 rpm shake speed

• Include buffer-only channel as reference for baseline correction

• Regenerate sensor surface between cycles using 10 mM glycine pH 2.0

• Analyze data using appropriate binding models to determine kon, koff, and KD values

This approach provides quantitative binding parameters that can help understand the affinity and specificity of the antibody-antigen interaction .

What strategies can be employed when NIFU5 Antibody shows unexpected cross-reactivity?

If cross-reactivity is observed:

• Increase blocking stringency (try 5% BSA instead of milk, or vice versa)

• Optimize antibody concentration with titration experiments

• Increase washing steps and duration

• Use highly purified samples to reduce non-specific binding

• Perform pre-absorption with related proteins

• Validate results with genetic knockouts/knockdowns of NIFU5

• Consider peptide competition assays to confirm specificity

These approaches can help distinguish between specific and non-specific signals when working with polyclonal antibodies that may contain a heterogeneous mixture of antibodies with varying specificities .

How should batch-to-batch variability of NIFU5 Antibody be addressed in long-term research projects?

To mitigate the impact of batch-to-batch variability:

• Validate each new lot against a reference batch

• Compare activity curves in standardized ELISA titrations

• Assess specificity in Western blots with positive and negative controls

• Reserve a portion of the previous validated lot for comparative testing

• Document lot-specific optimal dilutions and working conditions

• Consider purchasing larger quantities of a single lot for critical long-term studies

This systematic approach helps maintain experimental reproducibility across different antibody lots, as demonstrated in quality control procedures for other research antibodies .

How can epitope mapping be performed to characterize the binding site of NIFU5 Antibody?

Epitope mapping can be conducted through:

• Peptide array analysis:

  • Synthesize overlapping peptides (15-20 amino acids) spanning the entire NIFU5 protein

  • Spot peptides onto membrane and probe with NIFU5 Antibody

  • Identify reactive peptides to determine the linear epitope region

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare deuterium uptake of NIFU5 protein alone versus NIFU5-antibody complex

  • Regions protected from exchange indicate antibody binding sites

• Mutagenesis approach:

  • Generate point mutations or deletion variants of NIFU5 protein

  • Test antibody binding to mutant proteins by ELISA or Western blot

  • Identify critical residues required for antibody recognition

This information is valuable for understanding antibody function and potential cross-reactivity with related proteins .

How can machine learning approaches improve antibody-antigen binding prediction for NIFU5 research?

Recent advances in machine learning can enhance antibody research:

• Library-on-library approaches can predict binding between NIFU5 antibodies and variant antigens

• Active learning strategies can significantly reduce the experimental burden by:

  • Starting with a small labeled dataset of binding interactions

  • Iteratively expanding the labeled dataset based on model uncertainty

  • Selecting the most informative experiments to perform next

Studies have shown that optimized active learning algorithms can reduce the number of required antigen mutant variants by up to 35% and accelerate the learning process compared to random sampling approaches .

What considerations should be made when using NIFU5 Antibody in plant immunohistochemistry studies?

For immunohistochemistry applications with NIFU5 Antibody:

• Tissue fixation optimization:

  • Compare different fixatives (4% paraformaldehyde, glutaraldehyde)

  • Optimize fixation time to balance antigen preservation and antibody accessibility

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0)

  • Enzymatic retrieval with proteinase K

• Signal amplification systems:

  • Tyramide signal amplification

  • ABC (avidin-biotin complex) amplification

• Controls specific to plant tissues:

  • NIFU5 knockout/knockdown plants as negative controls

  • Tissues with known high expression as positive controls

  • Omission of primary antibody

  • Pre-absorption with recombinant NIFU5 protein

These considerations address the unique challenges of plant tissue immunohistochemistry, including cell wall barriers and autofluorescence issues.

How might NIFU5 Antibody be utilized in studying plant-pathogen interactions?

NIFU5 Antibody could be valuable in investigating:

• Changes in NIFU5 expression and localization during pathogen infection

• Post-translational modifications of NIFU5 in response to biotic stress

• Protein-protein interactions that may form or dissolve during immune responses

• Development of immunoassays to monitor NIFU5 as a potential biomarker for plant health

These applications would require validation of the antibody in various stress conditions and potentially developing new protocols specific to pathogen-infected tissues.

What considerations should be made when adapting NIFU5 Antibody for multiplex immunoassays?

For multiplex applications combining NIFU5 with other antibodies:

• Antibody compatibility testing:

  • Ensure no cross-reactivity between different primary antibodies

  • Validate specificity in the presence of multiple detection systems

• Optimization strategies:

  • Sequential incubation approaches versus simultaneous detection

  • Blocking optimization to minimize background in complex detection systems

  • Signal separation through fluorophore selection with minimal spectral overlap

• Validation controls:

  • Single-plex controls alongside multiplex experiments

  • Spike-in recovery tests to assess potential interference