NPRL3 Antibody, HRP conjugated

What is NPRL3 and what cellular functions does it have?

NPRL3 (Nitrogen permease regulator 3-like protein) is a 569 amino acid protein with a molecular weight of approximately 63.6 kDa that functions as a component of the GATOR1 complex . It plays a critical role as an inhibitor of the amino acid-sensing branch of the TORC1 pathway, strongly influencing GTP hydrolysis activity . NPRL3 is primarily localized in lysosomes and is widely expressed across multiple tissue types, including significant expression in red blood cells at both mRNA and protein levels .

The protein has been implicated in autophagy pathways and has several aliases including GATOR complex protein NPRL3, Alpha-globin regulatory element-containing gene protein, Protein CGTHBA, C16orf35, CGTHBA, and MARE . Research has associated the NPRL3 gene with epilepsy, suggesting its involvement in neurological functions . As a component of cellular signaling, NPRL3 participates in nutrient sensing mechanisms that regulate cellular metabolism and growth.

What are the specific characteristics of NPRL3 Antibody, HRP conjugated?

NPRL3 Antibody, HRP conjugated is a polyclonal antibody raised in rabbits using recombinant Human GATOR complex protein NPRL3 (amino acids 349-482) as the immunogen . This antibody has been purified using Protein G with >95% purity and is maintained in liquid form . The antibody is directly conjugated to horseradish peroxidase (HRP), which facilitates direct detection without requiring a secondary antibody in various immunoassays .

Key specifications include:

• Host species: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Immunogen: Recombinant Human GATOR complex protein NPRL3 (amino acids 349-482)

• Reactivity: Human

• Applications: Primarily ELISA, with potential for Western Blot applications

• Storage buffer: 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4

What are the recommended storage conditions for NPRL3 Antibody, HRP conjugated?

Proper storage of NPRL3 Antibody, HRP conjugated is essential for maintaining its activity and specificity. Upon receipt, the antibody should be stored at -20°C or -80°C . It's crucial to avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of enzymatic activity of the HRP conjugate .

For short-term storage (less than one month), the antibody can be stored at 4°C, but this is not recommended for long-term preservation. When handling the antibody, researchers should:

• Aliquot the antibody into smaller volumes upon first thawing to minimize freeze-thaw cycles

• Thaw aliquots slowly at 4°C or on ice rather than at room temperature

• Avoid exposure to light, particularly with HRP-conjugated antibodies, as light can reduce HRP activity

• Return the antibody to appropriate storage conditions immediately after use

• Monitor expiration dates, as HRP conjugated antibodies typically have shorter shelf lives than unconjugated antibodies

How should I optimize ELISA protocols for NPRL3 Antibody, HRP conjugated?

When optimizing ELISA protocols with NPRL3 Antibody, HRP conjugated, several methodological considerations can improve sensitivity and specificity:

• Antibody titration: Perform an initial titration experiment using a dilution series (typically 1:500 to 1:5000) to determine the optimal antibody concentration that maximizes specific signal while minimizing background .

• Blocking optimization: Test different blocking reagents (5% BSA, 5% non-fat milk, commercial blocking buffers) to identify which provides the lowest background with NPRL3 Antibody.

• Incubation conditions:

  • Temperature: Test both room temperature and 4°C incubations

  • Duration: Compare 1-hour, 2-hour, and overnight incubations

  • Washing buffer: PBS-T (0.05% Tween-20 in PBS) at pH 7.4 is typically effective

• Substrate selection: HRP-conjugated antibodies work with multiple substrates:

  • TMB (3,3',5,5'-Tetramethylbenzidine) - highest sensitivity, blue color changing to yellow upon stopping

  • ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) - green color

  • OPD (o-Phenylenediamine dihydrochloride) - orange-brown color

• Signal development time: Monitor the color development closely and optimize the timing before adding stop solution to achieve maximum sensitivity without saturation.

A typical optimization matrix might look like this:

What controls should be included in experiments using NPRL3 Antibody, HRP conjugated?

Rigorous experimental design requires appropriate controls when working with NPRL3 Antibody, HRP conjugated:

• Positive control: Include cell lines or tissues known to express NPRL3, such as red blood cells or cell lines with confirmed NPRL3 expression .

• Negative control:

  • No primary antibody control (to assess non-specific binding of detection systems)

  • Isotype control (rabbit IgG, HRP-conjugated at the same concentration)

  • Tissues or cells with NPRL3 knockdown/knockout (if available)

• Blocking peptide control: Competition assay with the immunizing peptide (amino acids 349-482) to confirm antibody specificity .

• Antibody validation controls:

  • siRNA knockdown of NPRL3 to confirm antibody specificity

  • Recombinant NPRL3 protein as a standard in ELISA

  • Western blot showing a single band at the expected molecular weight (63.6 kDa)

• Technical controls:

  • Internal loading control for normalization (housekeeping protein)

  • Standard curve using recombinant NPRL3 protein

  • Replicate samples to assess technical variability

How can I troubleshoot weak or inconsistent signals when using NPRL3 Antibody, HRP conjugated?

When encountering weak or inconsistent signals with NPRL3 Antibody, HRP conjugated, consider the following methodological troubleshooting steps:

• Antibody activity issues:

  • Check storage conditions and expiration date

  • Test a fresh aliquot that hasn't undergone multiple freeze-thaw cycles

  • Verify HRP conjugate activity using a direct ELISA against the immunogen

• Protocol optimization:

  • Increase antibody concentration (reduce dilution factor)

  • Extend incubation time or change temperature

  • Enhance detection sensitivity by using a more sensitive substrate system

• Sample preparation issues:

  • Ensure proper protein extraction with protease inhibitors

  • Verify protein quantification and loading

  • Check sample integrity (avoid protein degradation)

• Signal enhancement strategies:

  • Use signal amplification systems compatible with HRP

  • Optimize blocking to reduce background and improve signal-to-noise ratio

  • Consider using enhancers such as 0.1% Triton X-100 in antibody diluent

• Technical considerations:

  • Verify that the detection instrument is functioning properly

  • Check that the substrate is fresh and properly prepared

  • Ensure appropriate wavelength settings on plate readers

How can NPRL3 Antibody be used to study the mTOR signaling pathway?

NPRL3 functions as a component of the GATOR1 complex that inhibits the amino acid-sensing branch of the TORC1 pathway . Researchers can use NPRL3 Antibody, HRP conjugated to investigate this pathway through several approaches:

• Co-immunoprecipitation studies:

  • Using NPRL3 antibody to pull down interaction partners within the GATOR1 complex

  • Western blotting for mTOR pathway components to identify interactions

  • Analyzing how nutrient conditions affect NPRL3 interactions

• Pathway activation analysis:

  • Monitoring NPRL3 expression and localization in response to amino acid starvation/stimulation

  • Correlating NPRL3 levels with downstream mTOR targets such as phosphorylated S6 ribosomal protein

  • Comparing wild-type vs. NPRL3-depleted cells for differences in mTOR signaling dynamics

• Quantitative pathway analysis:

  • Using ELISA with NPRL3 Antibody to quantify protein levels in different cellular states

  • Developing multiplexed assays to simultaneously detect NPRL3 and other pathway components

  • Creating activation state-specific assays to monitor NPRL3 function in real-time

• Structure-function studies:

  • Analyzing NPRL3 domains using truncated constructs

  • Identifying critical regions for GATOR1 complex formation

  • Mapping amino acid-sensing domains using site-directed mutagenesis

A typical experimental design might include:

What methodologies can be used to investigate NPRL3's role in autophagy pathways?

NPRL3's involvement in autophagy pathways can be investigated using NPRL3 Antibody, HRP conjugated through the following methodological approaches:

• Autophagy flux assessment:

  • Monitoring LC3-I to LC3-II conversion in relation to NPRL3 expression

  • Analyzing p62/SQSTM1 degradation as an autophagy marker

  • Using tandem fluorescent LC3 reporters to distinguish autophagosome formation from lysosomal fusion

• NPRL3 localization studies:

  • Tracking NPRL3 co-localization with autophagosome markers

  • Examining NPRL3 recruitment to lysosomes during autophagy induction

  • Visualizing NPRL3 dynamics during amino acid starvation and recovery

• Genetic manipulation approaches:

  • Creating NPRL3 knockout/knockdown models to assess autophagy impairment

  • Complementing with wild-type or mutant NPRL3 to identify critical domains

  • Using inducible systems to study acute vs. chronic effects of NPRL3 depletion

• Pharmacological intervention:

  • Treating cells with autophagy inducers (rapamycin, Torin1) or inhibitors (chloroquine, bafilomycin A1)

  • Measuring NPRL3 expression changes using ELISA with HRP-conjugated antibody

  • Assessing pathway crosstalk through combinatorial drug treatments

Key experimental readouts might include:

How can NPRL3 Antibody be used to study its association with epilepsy?

The NPRL3 gene has been associated with epilepsy , and researchers can use NPRL3 Antibody, HRP conjugated to investigate this connection through several methodological approaches:

• Clinical sample analysis:

  • Comparing NPRL3 protein levels in epilepsy patients vs. controls

  • Correlating NPRL3 expression with epilepsy subtypes or severity

  • Examining NPRL3 in surgically resected epileptogenic tissues

• Neuronal excitability studies:

  • Investigating NPRL3 expression in response to seizure activity in neuronal cultures

  • Correlating NPRL3 levels with electrophysiological measurements

  • Examining NPRL3's relationship with ion channels implicated in epilepsy

• Animal model investigations:

  • Studying NPRL3 expression in rodent models of epilepsy

  • Analyzing temporal changes in NPRL3 levels before, during, and after seizures

  • Testing whether NPRL3 modulation affects seizure threshold or severity

• Mechanistic studies:

  • Investigating how NPRL3-mediated mTOR regulation affects neuronal hyperexcitability

  • Examining NPRL3's impact on synaptic plasticity and network formation

  • Studying potential links between NPRL3 and known epilepsy-associated pathways

Research questions might be structured as:

Technical Considerations and Method Validation

To quantify NPRL3 expression across different tissues using NPRL3 Antibody, HRP conjugated, researchers can employ several methodological approaches:

• Quantitative ELISA:

  • Developing a sandwich ELISA using captured and detection antibodies for NPRL3

  • Creating standard curves with recombinant NPRL3 protein

  • Normalizing to total protein content or housekeeping proteins

• Western blot quantification:

  • Using HRP-conjugated NPRL3 antibody for direct detection

  • Employing digital imaging and densitometry software

  • Including standard curves of recombinant protein for absolute quantification

• Tissue microarray analysis:

  • Creating arrays with multiple tissue samples

  • Using NPRL3 Antibody for immunohistochemistry

  • Employing digital pathology tools for quantitative analysis

• Multiplex assays:

  • Developing bead-based multiplex assays for simultaneous detection of NPRL3 and related proteins

  • Using different fluorophores or enzyme reporters for multiple targets

  • Creating tissue-specific protein expression profiles

A systematic tissue expression profile might be presented as:

What are the potential pitfalls in interpreting NPRL3 expression data using HRP-conjugated antibodies?

When interpreting NPRL3 expression data using HRP-conjugated antibodies, researchers should be aware of several methodological pitfalls:

• Technical limitations:

  • HRP activity can be affected by storage conditions and repeated freeze-thaw cycles

  • Endogenous peroxidase activity in tissues can lead to false-positive results

  • Signal saturation can make quantitative comparisons unreliable

• Antibody specificity issues:

  • Cross-reactivity with related proteins may occur despite validation

  • Post-translational modifications of NPRL3 might mask or create epitopes

  • Different isoforms of NPRL3 may not be equally detected

• Sample preparation artifacts:

  • Fixation methods can affect epitope accessibility

  • Protein extraction protocols may not equally recover NPRL3 from different subcellular compartments

  • Protease activity during sample handling can create truncated forms

• Interpretation challenges:

  • Correlating protein levels with functional significance requires careful analysis

  • Background levels can vary between tissues, affecting signal-to-noise ratios

  • Comparison between different experimental batches requires proper normalization

• Methodological considerations:

  • Direct HRP conjugation might affect antibody binding properties compared to unconjugated versions

  • Detection sensitivity varies between applications (ELISA vs. Western blot)

  • Quantitative comparisons require careful standard curve preparation

Potential solutions for these pitfalls include: