NUDC (Ab-326) Antibody

What is NudC and what are its primary biological functions?

NudC (Nuclear distribution protein C homolog) is a highly conserved protein initially identified for its role in cytoplasmic dynein-mediated nuclear migration in Aspergillus nidulans. In mammals, NudC performs multiple critical biological functions:

• Neuronal development: NudC is required for neuronal migration during neocorticogenesis and plays a specific role in apical nuclear migration in radial glial progenitor cells. It interacts genetically with Aspergillus NudF and physically with its mammalian orthologue Lis1, which is crucial for nuclear and neuronal migration during brain development .

• Cell division regulation: NudC is necessary for the correct formation of mitotic spindles, chromosome separation during mitosis, cytokinesis, and cell proliferation .

• Cytoskeletal organization: NudC regulates microtubule stability, with research demonstrating that loss of NudC in axon terminals results in reduced dynamic microtubule plus ends and decreased EB3 comet number .

• Protein interactions: NudC associates with multiple proteins including Lis1, PSME3, and CCP6, functioning in various cellular processes including cell migration and adhesion .

Recent studies have also revealed that NudC displays both cytoplasmic and nuclear localization, with strong enrichment at the midbody during cytokinesis .

What epitope does the NUDC (Ab-326) Antibody recognize?

The NUDC (Ab-326) Antibody specifically recognizes an epitope centered around the serine 326 phosphorylation site of human NudC. The immunogen used to generate this antibody is a synthesized non-phosphopeptide derived from human NudC with the sequence D-F-S(p)-K-A, where S(p) represents serine 326 .

The antibody is designed to detect endogenous levels of total NudC protein, not exclusively the phosphorylated form, making it suitable for detecting NudC regardless of its phosphorylation state. This feature makes the antibody valuable for studying NudC in various experimental conditions and cellular contexts .

What applications has NUDC (Ab-326) Antibody been validated for?

NUDC (Ab-326) Antibody has been primarily validated for Western blotting (WB) applications. According to product specifications, the recommended dilution range for Western blotting is 1:500 to 1:3000 .

Scientific validation data demonstrates successful detection of endogenous NudC in rat brain cell extracts via Western blot, producing a band at approximately 38 kDa, which corresponds to the calculated molecular weight of NudC .

While Western blotting is the primary validated application, research studies investigating NudC suggest that similar antibodies have been successfully employed in other applications such as:

• Immunofluorescence microscopy

• Co-immunoprecipitation

• Proximity ligation assay (PLA)

Researchers should perform their own validation when using this antibody for applications beyond Western blotting.

What species reactivity does NUDC (Ab-326) Antibody demonstrate?

According to the product specifications, NUDC (Ab-326) Antibody has been validated to react with:

• Human (Hu)

• Rat (Rt)

Additional product information indicates potential reactivity with mouse samples, though this may require additional validation by individual researchers . The multi-species reactivity is likely due to the high conservation of the NudC protein sequence across mammalian species, particularly in the region surrounding serine 326 (D-F-S-K-A) that was used as the immunogen .

What are the optimal storage conditions for NUDC (Ab-326) Antibody?

For optimal stability and performance of NUDC (Ab-326) Antibody, the following storage guidelines should be followed:

Unconjugated antibody:

• Store at -20°C

• Supplied in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol

• Avoid repeated freeze-thaw cycles

Conjugated antibody forms:

• Store at 4°C in the dark for up to 6 months

• Formulated in 0.01M Sodium Phosphate, 0.25M NaCl, pH 7.6, 5mg/ml Bovine Serum Albumin, 0.02% Sodium Azide

For long-term storage stability, prepare small aliquots to minimize freeze-thaw cycles, as each cycle can potentially reduce antibody activity. When handling the antibody, use sterile techniques and avoid contamination.

What is the optimal Western blotting protocol for NUDC (Ab-326) Antibody?

Based on validated methodologies, here is an optimized Western blotting protocol for NUDC (Ab-326) Antibody:

Sample Preparation:

• Harvest cells or tissues (rat brain extracts work well as positive controls) and lyse in RIPA buffer containing protease and phosphatase inhibitors

• Quantify protein concentration using BCA or Bradford assay

• Mix samples with 4× Laemmli buffer and heat at 95°C for 5 minutes

• Load 10-30 μg protein per lane on a 10-12% polyacrylamide gel (optimal for the 38 kDa NudC protein)

SDS-PAGE and Transfer:

• Run gel at 100-120V until dye front reaches bottom

• Transfer to PVDF or nitrocellulose membrane (100V for 1 hour or 30V overnight at 4°C)

• Verify transfer efficiency with Ponceau S staining

Immunoblotting:

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

• Dilute NUDC (Ab-326) Antibody 1:500 to 1:3000 in 5% BSA in TBST

• Incubate overnight at 4°C with gentle rocking

• Wash 3-4 times with TBST, 5-10 minutes each

• Incubate with HRP-conjugated anti-rabbit IgG at 1:5000-1:10000 dilution for 1 hour at room temperature

• Wash 3-4 times with TBST, 5-10 minutes each

• Apply ECL substrate and expose to film or digital imager

Special considerations:

• For phosphorylation studies, include additional phosphatase inhibitors (PhosSTOP or equivalent)

• For detecting nuclear NudC, consider separate cytoplasmic and nuclear fractionation

• For studying NudC in the midbody, synchronize cells in cytokinesis (e.g., double thymidine block and release)

What conjugated versions of NUDC (Ab-326) Antibody are available and how do they compare?

NUDC (Ab-326) Antibody is available in multiple conjugated forms for various imaging applications. Below is a comparative analysis:

Available conjugates:

• Biotin

• Alexa Fluor dyes: AF350, AF405, AF488, AF555, AF594, AF647, AF680, AF750

Application-specific recommendations:

• Multiple protein co-localization studies:

  • Choose spectrally separated conjugates (e.g., AF488, AF594, AF647)

  • Consider AF350 if using DAPI for nuclear counterstaining

• Fixed tissue immunohistochemistry:

  • AF594, AF647, or AF680 for better tissue penetration and lower background

  • Biotin conjugate with streptavidin amplification for weak signals

• Super-resolution microscopy:

  • AF647 is excellent for STORM/PALM techniques

  • AF488, AF555 work well for STED microscopy

How can NUDC (Ab-326) Antibody be used to study neuronal migration?

NUDC (Ab-326) Antibody can be employed in several experimental approaches to investigate neuronal migration:

In vivo neuronal migration studies:
Research has demonstrated that NudC is required for neuronal migration during neocorticogenesis and plays a specific role in apical nuclear migration in radial glial progenitor cells . To study this:

• Combined with in utero electroporation:

  • Electroporate embryonic brain tissue with NudC shRNA constructs along with GFP markers

  • Use NUDC (Ab-326) Antibody to verify knockdown efficiency via Western blotting

  • Assess neuronal positioning in developing cortex using immunohistochemistry

• Immunohistochemistry of brain sections:

  • Use NUDC (Ab-326) Antibody on brain tissue sections at different developmental stages

  • Combine with markers for migrating neurons (Dcx, NeuN)

  • Analyze co-localization with Lis1, a known NudC interactor crucial for neuronal migration

Mechanistic studies:

• Co-immunoprecipitation with Lis1:

  • Use NUDC (Ab-326) Antibody in co-IP experiments to study the interaction between NudC and Lis1

  • Compare wild-type versus migrating neurons

• Proximity Ligation Assay (PLA):

  • Combine NUDC (Ab-326) Antibody with anti-Lis1 antibodies

  • Visualize NudC-Lis1 interactions in intact neuronal cells

  • Quantify interaction frequency during different migration phases

When using the antibody for these applications, appropriate controls should be included, including positive controls (rat brain extracts), negative controls (NudC knockdown samples), and isotype controls (rabbit IgG at the same concentration) .

How can NUDC (Ab-326) Antibody be used to investigate NudC's role in cell division?

NUDC (Ab-326) Antibody can be employed in multiple experimental approaches to investigate NudC's crucial role in cell division:

Immunofluorescence microscopy of dividing cells:

• Culture cells on coverslips and fix at different stages of mitosis

• Perform immunostaining with NUDC (Ab-326) Antibody (starting dilution 1:200)

• Co-stain with markers for:

  • Mitotic spindles (α-tubulin)

  • Centrosomes (γ-tubulin)

  • Midbody (Aurora B)

  • DNA (DAPI)

• Analyze NudC localization throughout mitosis

Research findings have shown that NudC strongly localizes to the midbody during cytokinesis, suggesting its role in regulating this process .

Functional studies:

• Knockdown NudC using siRNA/shRNA (verify using NUDC (Ab-326) Antibody)

• Assess mitotic defects:

  • Spindle formation abnormalities

  • Chromosome segregation errors

  • Cytokinesis failures

  • Multinucleation

Post-translational modification analysis:

• Immunoprecipitate NudC using NUDC (Ab-326) Antibody from synchronized cells

• Perform Western blot analysis with phospho-specific antibodies

• Consider mass spectrometry analysis to identify mitosis-specific modifications

NudC function during mitosis is regulated by phosphorylation by several mitotic kinases (Cdk, Plk1, and Aurora B). Since NUDC (Ab-326) Antibody targets a region around serine 326, it may be particularly useful for studying phosphorylation-dependent regulation of NudC during cell division .

How does NudC interact with bacterial Nudix hydrolase NudC and is there any confusion in the literature?

It's important to clarify that mammalian NudC (Nuclear distribution protein C homolog) and bacterial NudC (a Nudix hydrolase) are entirely different proteins despite sharing the same name, which has caused some confusion in the literature:

Mammalian NudC:

• A 38 kDa protein involved in nuclear migration, neuronal development, and cell division

• Interacts with Lis1 and dynein complex

• Target of the NUDC (Ab-326) Antibody discussed in this FAQ

• Encoded by the NUDC gene (human Gene ID: 10726, UniProt: Q9Y266)

Bacterial NudC:

• A Nudix hydrolase enzyme involved in RNA processing, specifically NAD decapping

• Structure revealed by crystallography shows it forms a homodimer with NAD binding at the dimer interface

• Bacterial enzyme with no sequence homology to mammalian NudC

• Described in detail in research focusing on bacterial RNA metabolism

When researching NudC, scientists should:

• Pay careful attention to which NudC protein is being referenced

• Note the molecular weight (38 kDa for mammalian vs. different for bacterial)

• Consider the biological context (eukaryotic vs. prokaryotic)

• Be aware of this naming conflict when searching literature databases

The NUDC (Ab-326) Antibody specifically recognizes mammalian NudC protein and not the bacterial Nudix hydrolase .

What are the best approaches for studying NudC-protein interactions using NUDC (Ab-326) Antibody?

Several complementary approaches can be used with NUDC (Ab-326) Antibody to study NudC protein interactions:

1. Proximity Ligation Assay (PLA):
This technique has been successfully used to detect NudC interactions in research studies. PLA allows visualization of protein interactions within cells with high sensitivity.

Protocol outline:

• Fix cells on coverslips (4% paraformaldehyde, 10 min)

• Permeabilize (0.2% Triton X-100, 10 min)

• Block (1% BSA, 1 hour)

• Incubate with NUDC (Ab-326) Antibody and antibody against potential interacting protein

• Follow manufacturer's instructions for PLA probes and detection reagents

• Counterstain nuclei with DAPI

• Image using confocal microscopy

Research findings using PLA showed:

• NudC-PSME3 interaction was detected primarily in the nucleus

• NudC-CCP6 interaction displayed a cytoplasmic pattern in the perinuclear region

2. Co-immunoprecipitation with optimization:

• Consider crosslinking approaches for transient interactions

• For nuclear interactions (e.g., with PSME3), use nuclear extraction protocols

• For cytoskeletal interactions, use buffers that preserve these structures

3. Key interaction partners to investigate:
Based on research findings:

• Lis1: Critical for neuronal migration

• PSME3: Regulates migration and differentiation of myoblasts

• CCP6: Associated with NudC in the midbody during cytokinesis

• Dynein components: NudC regulates dynein-mediated processes

Important considerations:

• NudC contains multiple domains that may mediate different interactions

• Phosphorylation at serine 326 (the target region of NUDC (Ab-326) Antibody) may affect certain interactions

• Some interactions (e.g., with CCP6) may be transient and difficult to detect by standard co-IP

How can NUDC (Ab-326) Antibody be used to study the interaction between NudC and PSME3?

The interaction between NudC and PSME3 (Proteasome activator complex subunit 3) represents an important area of research. NUDC (Ab-326) Antibody can be employed in multiple complementary approaches to study this specific interaction:

1. Proximity Ligation Assay (PLA):
Research findings demonstrated successful use of PLA to detect NudC-PSME3 interaction with key observations:

• PLA revealed NudC-PSME3 association specifically within the nucleus

• This was unexpected as NudC was traditionally considered predominantly cytoplasmic

• The interaction has implications for cell migration and differentiation

Protocol:

• Grow cells on coverslips and fix with 4% paraformaldehyde

• Permeabilize with 0.2% Triton X-100

• Block with 1% BSA

• Incubate with NUDC (Ab-326) Antibody and anti-PSME3 antibody

• Proceed with PLA according to manufacturer's protocol

• Image using confocal microscopy

2. Functional studies:
Research findings showed that:

• PSME3 knockdown increased cell migration rate

• This was linked to altered levels of cell adhesion proteins

• PSME3 interacts with NudC to regulate both migration and adhesion

Experimental approach:

• Perform PSME3 knockdown and analyze effects on:

  • NudC localization using NUDC (Ab-326) Antibody via immunofluorescence

  • NudC protein levels by Western blot

  • Cell migration using scratch assays

• Examine protein relationships:

  • Assess co-localization of NudC and PSME3 during different stages of myoblast differentiation

  • Compare wild-type vs. PSME3-depleted cells

  • Analyze effects on common downstream targets including cell adhesion proteins (MYADM, ITGB3)

What are essential controls when using NUDC (Ab-326) Antibody?

When using NUDC (Ab-326) Antibody, comprehensive controls are essential to ensure reliable and interpretable results. Here are the recommended control experiments for different applications:

For Western Blotting:

• Positive control:

  • Use samples known to express NudC (e.g., rat brain extracts as shown in validation data)

  • Include recombinant NudC protein if available

• Negative control:

  • NudC knockdown samples (siRNA/shRNA treated)

  • Secondary antibody only (omit primary antibody)

  • Isotype control antibody (rabbit IgG at same concentration)

• Loading control:

  • Probe for housekeeping proteins (β-actin, GAPDH)

  • Use total protein staining methods (Ponceau S)

For Immunofluorescence:

• Antibody specificity controls:

  • Secondary antibody only

  • Isotype control (rabbit IgG)

  • Pre-absorption with immunizing peptide (if available)

• Expression verification:

  • NudC knockdown samples

  • Compare staining pattern with published results (cytoplasmic, nuclear, and midbody localization)

For Proximity Ligation Assay (PLA):

• Single antibody controls:

  • Perform PLA with only NUDC (Ab-326) Antibody

  • Perform PLA with only the interacting protein antibody

• Negative interaction control:

  • Use antibodies against proteins not expected to interact with NudC

Based on research findings, researchers should be particularly attentive to:

• NudC's multiple localizations (cytoplasmic, nuclear, midbody)

• Potential phosphorylation effects on detection

• Protein-protein interactions that may be cell-cycle dependent or transient

How can non-specific binding be addressed when using NUDC (Ab-326) Antibody?

Non-specific binding can interfere with accurate interpretation of results. Here's a troubleshooting guide for addressing non-specific binding with NUDC (Ab-326) Antibody:

Common issues in Western blot and solutions:

Immunofluorescence troubleshooting:

Validation strategies:

• Peptide competition assay:

  • Pre-incubate NUDC (Ab-326) Antibody with immunizing peptide (D-F-S-K-A)

  • Perform Western blot or immunostaining in parallel with non-blocked antibody

  • Specific signal should be reduced/eliminated

• Knockdown validation:

  • Perform Western blot on NudC-depleted samples

  • Specific bands should be reduced/absent

Research findings indicate that NudC has multiple localizations (cytoplasmic, nuclear, midbody) depending on cell type and cell cycle stage. When troubleshooting, consider that legitimate signal might appear in these various locations .

How does phosphorylation at serine 326 affect antibody binding and experimental design?

The NUDC (Ab-326) Antibody was raised against a synthetic non-phosphopeptide derived from human NudC around the phosphorylation site of serine 326 (D-F-S(p)-K-A). Understanding this relationship is crucial for experimental design:

Antibody specificity and phosphorylation:

• The antibody detects endogenous levels of total NudC protein, indicating it recognizes both phosphorylated and non-phosphorylated forms

• Since the immunogen was a non-phosphopeptide, the antibody likely has higher affinity for the non-phosphorylated form

Experimental implications:

• Cell cycle studies:
  Research shows NudC is phosphorylated by mitotic kinases (Cdk, Plk1, and Aurora B). When studying NudC during mitosis:

  • Compare staining patterns between interphase and mitotic cells

  • Use phosphatase treatments on parallel samples to determine if phosphorylation affects detection

  • Consider complementing with phospho-specific antibodies if available

• Kinase inhibitor experiments:

  • If treating cells with inhibitors of kinases known to target NudC, monitor whether this affects detection intensity

• Immunoprecipitation considerations:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status

  • Consider that phosphorylation may affect protein-protein interactions

The serine 326 phosphorylation site appears significant, as evidenced by its selection as the target region for antibody generation. Research indicates that NudC function is regulated by phosphorylation, particularly during mitosis and in neuronal migration. The phosphorylation status may affect NudC's interactions with partners like Lis1, dynein components, or PSME3 .

How does NudC regulate microtubule dynamics and how can this be studied?

Recent studies have revealed an important role for NudC in regulating microtubule dynamics, particularly in axon terminals. NUDC (Ab-326) Antibody can be employed to investigate this function:

Key research findings:

• NudC mutants show reduced microtubule growth dynamics in axon terminals

• Analysis of EB3-GFP comets (microtubule plus end binding protein) demonstrated a strong reduction in comet number with loss of NudC

• This suggests NudC is essential for maintaining dynamic microtubule plus ends

Experimental approaches:

• Live imaging of microtubule dynamics:

  • Transfect cells with EB3-GFP to visualize growing microtubule plus ends

  • Knockdown NudC using siRNA (validated by Western blot with NUDC (Ab-326) Antibody)

  • Perform time-lapse microscopy to track EB3 comets

  • Quantify microtubule growth parameters (growth rate, catastrophe frequency, etc.)

• Microtubule stability assays:

  • Treat cells with microtubule-destabilizing drugs

  • Compare sensitivity between control and NudC-depleted cells

  • Analyze microtubule regrowth after cold-induced depolymerization

• NudC-Lis1 interaction analysis:

  • Use NUDC (Ab-326) Antibody in PLA or co-IP studies to examine NudC-Lis1 interaction

  • Research supports a model where local NudC-Lis1 modulation of the dynein motor is critical for microtubule stability regulation

• Rescue experiments:

  • Perform NudC knockdown followed by re-expression of wild-type or mutant NudC

  • Use NUDC (Ab-326) Antibody to verify expression levels

  • Analyze microtubule dynamics to identify critical domains/residues

This research direction is particularly relevant for understanding neuronal development and axon maintenance, as proper microtubule dynamics are essential for these processes.

How is NudC involved in post-translational modifications and protein stability?

Emerging research suggests NudC plays important roles in post-translational modifications and protein stability regulation. NUDC (Ab-326) Antibody can be valuable in investigating these functions:

Key research findings:

• NudC interacts with CCP6 (Cytosolic carboxypeptidase 6), a deglutamylating enzyme involved in protein modification

• NudC associates with PSME3 (also known as REGγ), a proteasome activator, suggesting a potential role in protein degradation pathways

• NudC's function is regulated by various post-translational modifications, including phosphorylation and potentially others

Experimental approaches:

• Studying NudC-CCP6 interaction:

  • Use NUDC (Ab-326) Antibody in PLA studies to visualize this interaction

  • Investigate whether NudC itself is subject to glutamylation

  • Examine if CCP6 activity affects NudC stability or function

• NudC-PSME3 pathway analysis:

  • Examine NudC stability in PSME3-depleted cells using NUDC (Ab-326) Antibody

  • Investigate whether proteasome inhibitors affect NudC levels

  • Identify potential degradation signals in NudC

• NudC modification mapping:

  • Immunoprecipitate NudC using NUDC (Ab-326) Antibody

  • Perform mass spectrometry to identify post-translational modifications

  • Generate modification-specific mutants to test functional significance

Research has suggested: "Hence, it would be interesting to thoroughly assess whether glutamylation would also play a role in the finetuning of NudC functioning in these key cellular processes" . This represents an emerging area where NUDC (Ab-326) Antibody could provide valuable insights.

What are the latest developments in NudC research where NUDC (Ab-326) Antibody could be applied?

Recent developments in NudC research present exciting opportunities for applications of NUDC (Ab-326) Antibody:

• Cell migration and adhesion regulation:

  • Recent studies have revealed that NudC interacts with PSME3 to regulate cell migration and adhesion protein expression

  • Depletion of PSME3 increases migration rate and affects levels of key adhesion proteins including MYADM and ITGB3

  • NUDC (Ab-326) Antibody could be used to investigate how NudC's role in this pathway is regulated

• Myoblast differentiation:

  • NudC has been implicated in muscle differentiation processes

  • NUDC (Ab-326) Antibody could help track NudC expression and localization changes during myoblast differentiation

  • This may provide insights into muscle development disorders

• Nuclear vs. cytoplasmic functions:

  • While traditionally considered cytoplasmic, recent research shows NudC also has nuclear functions

  • NUDC (Ab-326) Antibody could be used to investigate what regulates NudC's nuclear localization and its nuclear-specific interactions

• Midbody localization during cytokinesis:

  • NudC strongly localizes to the midbody during cell division

  • NUDC (Ab-326) Antibody could help elucidate NudC's role in cytokinesis regulation

  • This may have implications for understanding cell division defects

• Post-translational modification regulation:

  • NudC is subject to various modifications including phosphorylation

  • Novel research suggests potential involvement in glutamylation pathways via CCP6 interaction

  • NUDC (Ab-326) Antibody could help investigate how these modifications affect NudC function