NRBF2 Antibody

What is NRBF2 and why is it important to study?

NRBF2 is a 40 kDa Beclin 1-binding protein that functions as a key component and regulator of the phosphatidylinositol 3-kinase (PtdIns3K) complex . The protein contains two discrete structural motifs: an N-terminal microtubule interaction and trafficking (MIT) domain and a C-terminal coiled coil domain (CCD) . NRBF2 is significant because it regulates autophagy induction, interacts with multiple proteins in the Beclin 1-Vps34 complexes (particularly associated with Atg14L), and plays crucial roles in neurodegenerative processes such as Alzheimer's disease through its involvement in APP-CTFs homeostasis . Recent research has also revealed NRBF2's importance in learning and memory processes, making it a valuable target for neuroscience research .

What types of samples can be analyzed using NRBF2 antibodies?

NRBF2 antibodies can be used to detect the protein in various sample types including:

• Brain tissue lysates (mouse brain lysates have been successfully used to study endogenous NRBF2)

• Hippocampal tissue (particularly relevant for memory and learning studies)

• Cell lines such as N2a mouse neuroblastoma cells expressing human Swedish mutant APP695 (N2S cells), HEK293 cells, and Chinese hamster ovary (CHO) cells

• Tissue from NRBF2 knockout and wild-type mice for comparative studies

The detection typically involves techniques such as immunoprecipitation, Western blotting, and immunofluorescence microscopy for co-localization studies .

How can I verify the specificity of an NRBF2 antibody?

To verify antibody specificity:

• Perform Western blot analysis comparing wild-type tissues/cells with NRBF2 knockout samples. The absence of the band in knockout samples confirms specificity .

• Use NRBF2 siRNA knockdown as a control—the signal should be significantly reduced in knockdown samples .

• Conduct immunoprecipitation experiments to confirm the antibody can pull down known NRBF2 interaction partners (Beclin 1, Vps34, Vps15, Atg14L, and UVRAG) .

• Perform peptide competition assays where pre-incubation with the immunizing peptide should abolish specific antibody binding.

• Compare results across multiple NRBF2 antibodies targeting different epitopes of the protein.

How can NRBF2 antibodies be used to study autophagy regulation?

NRBF2 antibodies are valuable tools for investigating autophagy mechanisms:

• Protein complex analysis: Use co-immunoprecipitation with anti-NRBF2 antibodies to pull down and analyze associated autophagy proteins including Vps34, Vps15, Atg14L, and UVRAG .

• Autophagosome formation: Employ immunofluorescence staining with NRBF2 antibodies alongside LC3 and WIPI2 antibodies to visualize and quantify NRBF2's role in phagophore and autophagosome formation .

• Lipid kinase activity assays: After immunoprecipitation with NRBF2 antibodies, conduct lipid kinase assays to measure the activity of associated Vps34 complexes, revealing how NRBF2 modulates PI3K activity essential for autophagy initiation .

• Autophagic flux assays: In experiments where NRBF2 is manipulated (knockout/overexpression), use NRBF2 antibodies alongside LC3B-II and SQSTM1/p62 antibodies to monitor changes in autophagic activity .

• Subcellular localization studies: Determine how NRBF2 redistributes under different conditions (e.g., nutrient starvation) using fractionation followed by immunoblotting or immunofluorescence microscopy .

What is the role of NRBF2 in neurodegenerative disease research and how can antibodies help investigate this?

NRBF2 antibodies are crucial for exploring the protein's role in neurodegenerative diseases:

• Alzheimer's disease mechanisms: NRBF2 antibodies can be used to monitor protein level changes in AD models, as NRBF2 is reduced in 5XFAD mice and affects APP-CTFs homeostasis .

• APP interaction studies: Co-immunoprecipitation with NRBF2 antibodies can confirm direct interactions with APP in both cell models and brain tissue from AD mouse models .

• Vesicular trafficking analysis: Immunostaining with NRBF2 antibodies alongside endosomal markers (e.g., RAB5) can track how NRBF2 influences the sorting of APP and APP-CTFs into endosomal intralumenal vesicles .

• Aβ production pathway: When combined with ELISA assays for Aβ detection, NRBF2 immunostaining or immunoblotting helps correlate NRBF2 levels with changes in Aβ production and accumulation .

• Memory deficit mechanisms: In behavioral studies of NRBF2 knockout mice, brain immunohistochemistry with NRBF2 antibodies helps correlate protein expression patterns with observed memory deficits .

How can NRBF2 antibodies be used in studying protein-protein interactions in autophagy complexes?

NRBF2 antibodies are instrumental for dissecting complex protein interaction networks:

• Sequential immunoprecipitation: Use anti-NRBF2 antibodies for first-round immunoprecipitation followed by elution and second-round precipitation with antibodies against suspected interaction partners to confirm direct versus indirect associations .

• Domain-specific interactions: Combine NRBF2 antibodies with domain deletion mutants (e.g., dMIT-CFP or dCCD-CFP) to map which protein domains are essential for specific protein-protein interactions .

• Proximity ligation assays: Employ NRBF2 antibodies in proximity ligation assays to visualize and quantify direct protein-protein interactions in situ with nanometer resolution.

• Mass spectrometry identification: Use anti-NRBF2 antibodies for affinity purification followed by mass spectrometry to identify novel interaction partners, as demonstrated in studies that identified Atg14L, Vps34, Vps15, and Beclin 1 as significant NRBF2 binding partners .

• Gel filtration analysis: Apply NRBF2 antibodies to detect the protein in different fractions after gel filtration to identify native protein complexes of varying molecular weights, revealing how NRBF2 associates with Atg14L and Beclin 1 in multiple complex assemblies .

What are the optimal conditions for using NRBF2 antibodies in Western blotting?

For successful Western blotting with NRBF2 antibodies:

• Sample preparation: Homogenize tissue in RIPA buffer supplemented with 1 mM PMSF, 1× protease inhibitor cocktail, and 1× phosphatase inhibitor cocktail. Centrifuge at 12,000g for 20 minutes at 4°C .

• Protein loading: Load approximately 20 μg of protein per sample on a 10% SDS-PAGE gel .

• Transfer conditions: Transfer proteins to PVDF membranes for optimal results .

• Blocking conditions: Block membranes in 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 hours at room temperature .

• Antibody dilution: Use NRBF2 rabbit monoclonal antibody at 1:1000 dilution (e.g., Proteintech, 24858-1-AP) .

• Incubation conditions: Incubate with primary antibody overnight at 4°C, followed by HRP-conjugated secondary antibodies (1:2000) in TBST with 1% BSA for 2 hours at room temperature .

• Detection method: Use enhanced chemiluminescence reagent and capture images with a digital imaging system such as ChemiDoc XRS+ .

How should I design experiments to study the effects of NRBF2 on autophagy using antibodies?

Design comprehensive experiments that address multiple aspects of autophagy:

• Comparative analysis models:

  • Use NRBF2 knockout models (generated via CRISPR-Cas9)

  • Create NRBF2 knockdown models using siRNA (achieving approximately 60% reduction)

  • Develop NRBF2 overexpression systems

  • Include appropriate controls (wild-type, scrambled siRNA, empty vector)

• Autophagy flux measurements:

  • Long-lived protein degradation assays using 3H Leucine release

  • LC3-I to LC3-II conversion by Western blot

  • SQSTM1/p62 accumulation or degradation

  • Autophagic vesicle quantification using WIPI2 and LC3 immunostaining

• Autophagy modulation:

  • Compare normal conditions versus nutrient starvation (HBSS buffer)

  • Include pharmacological modulators: rapamycin (inducer) and wortmannin (inhibitor)

  • Use Vps34 inhibitor SAR405 to distinguish autophagy-dependent from autophagy-independent functions

• Protein interaction validation:

  • Perform co-immunoprecipitation to confirm interactions with autophagy proteins

  • Validate with immunofluorescence co-localization studies

  • Conduct domain mapping using NRBF2 mutants missing either MIT domain or CCD

What controls should be included when using NRBF2 antibodies in immunoprecipitation experiments?

Include these essential controls for reliable immunoprecipitation results:

• Negative controls:

  • IgG control: Use non-specific IgG from the same species as the NRBF2 antibody

  • Lysate from NRBF2 knockout or knockdown cells/tissues

  • Pre-clearing samples with protein A/G beads alone to reduce non-specific binding

• Specificity controls:

  • Reciprocal immunoprecipitation (e.g., IP with anti-Beclin 1 antibody and blot for NRBF2, then vice versa)

  • Competition with excess immunizing peptide or recombinant NRBF2 protein

  • Comparison of results using multiple NRBF2 antibodies targeting different epitopes

• Validation controls:

  • Input control (5-10% of pre-IP lysate) to confirm presence of target proteins

  • Known interaction partners as positive controls (e.g., Beclin 1, Atg14L)

  • Non-interacting protein as negative control

• Experimental perturbation controls:

  • Atg14L knockdown to verify dependence of NRBF2-Beclin 1 interaction on Atg14L

  • NRBF2 knockdown to confirm antibody specificity and validate interaction results

How can I troubleshoot common issues with NRBF2 antibody detection?

Address these common challenges when working with NRBF2 antibodies:

• Weak or no signal in Western blots:

  • Increase antibody concentration or incubation time

  • Optimize protein extraction methods (RIPA buffer with protease inhibitors works well)

  • Try heat-denaturing samples at 100°C with 6× loading buffer for 10 minutes

  • Verify target expression level in your sample (NRBF2 levels vary by tissue type and can be reduced in some disease models)

• Multiple bands or non-specific binding:

  • Increase blocking stringency (5% BSA has been effective)

  • Optimize antibody dilution (1:1000 is recommended)

  • Perform peptide competition assay to identify specific bands

  • Use lysates from NRBF2 knockout samples as negative control

• Poor co-immunoprecipitation results:

  • Use gentler lysis buffers to preserve protein-protein interactions

  • Consider cross-linking proteins before lysis

  • Ensure antibody recognizes native protein (some antibodies only work on denatured proteins)

  • Check if the epitope is masked by protein interactions

• Inconsistent immunofluorescence staining:

  • Optimize fixation method (different proteins require different fixatives)

  • Test permeabilization conditions

  • Increase antibody concentration or incubation time

  • Use signal amplification methods if needed

How should I interpret changes in NRBF2 expression patterns in disease models?

Follow these guidelines for accurate interpretation:

• Expression level changes:

  • NRBF2 levels are reduced in 5XFAD mice (Alzheimer's model)

  • Compare with changes in other autophagy proteins (e.g., BECN1 is also reduced in AD models, while LC3B-II and SQSTM1/p62 levels are increased)

  • Normalize NRBF2 expression to housekeeping proteins (GAPDH has been successfully used)

  • Analyze time-dependent changes (e.g., Nrbf2 mRNA levels significantly increase at 6h and 12h after fear conditioning training)

• Functional correlations:

  • NRBF2 depletion impairs memory acquisition and subsequent short-term and long-term memory

  • NRBF2 overexpression reduces APP-CTFs and Aβ levels without affecting full-length APP or secreted APP fragments

  • NRBF2 knockout attenuates recruitment of APP and APP-CTFs into phagophores and endosomal intralumenal vesicles

• Pathway analysis:

  • Distinguish autophagy-dependent from autophagy-independent effects (e.g., use Vps34 inhibitor SAR405)

  • Consider parallel pathways (NRBF2 affects both autophagy and endosomal trafficking)

  • Analyze interactions with binding partners (MIT domain is critical for Atg14L interaction)

• Statistical approach:

  • Use appropriate statistical tests (unpaired Student's t-test for two groups, one-way ANOVA with Tukey's test for multiple groups)

  • Consider repeated two-way ANOVA with Bonferroni's post-hoc test for training results

  • Data should be considered statistically significant when P-values are <0.05

How can I quantitatively assess the impact of NRBF2 on autophagy using antibody-based techniques?

Implement these quantitative approaches:

• Autophagosome and phagophore quantification:

  • Count LC3-positive puncta (autophagosomes) and WIPI2-positive puncta (phagophores) per cell

  • Compare counts between wild-type, NRBF2 knockout, and rescue conditions

  • Analyze under different conditions: normal, rapamycin treatment, and nutrient withdrawal

• Autophagic flux measurement:

  • Calculate the ratio of LC3-II/LC3-I with and without lysosomal inhibitors

  • Measure long-lived protein degradation using 3H Leucine release assays

  • Compare results with and without wortmannin to determine autophagy-specific effects

• Protein complex activity:

  • Quantify lipid kinase activity from immunoprecipitated complexes

  • Determine Vps34/Vps15 protein levels in NRBF2 knockout versus rescue conditions

  • Monitor how MIT domain deletion affects complex formation and activity

• Image analysis approaches:

  • Use ImageJ software for optical density quantification of Western blot bands

  • Employ colocalization analysis to quantify NRBF2 overlap with autophagy markers

  • Perform time-lapse imaging to track dynamics of NRBF2-positive structures

• Data representation:

  • Present data as mean ± SEM with individual data points (dots) representing single mice or independent experiments

  • Ensure biological replication of at least three times for each experiment

  • Use appropriate statistical analyses to determine significance of observed differences