NAM8 Antibody

What is NAM8 Antibody and what are its primary research applications?

NAM8 Antibody is a polyclonal antibody raised in rabbits against recombinant Saccharomyces cerevisiae (strain ATCC 204508/S288c) NAM8 protein. It specifically recognizes the NAM8 protein (UniProt ID: Q00539), which functions as an RNA-binding component involved in mRNA splicing and processing in yeast. The primary research applications include Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB), making it valuable for detecting and quantifying NAM8 protein expression levels in experimental systems .

NAM8 antibody enables researchers to investigate RNA metabolism, pre-mRNA splicing mechanisms, and cellular stress responses in Saccharomyces cerevisiae. The specificity of this antibody for the yeast NAM8 protein makes it particularly valuable for studies focused on fungal molecular biology and comparative genomics.

What is the molecular basis for NAM8 protein recognition by this antibody?

The NAM8 antibody has been developed to recognize epitopes on the recombinant NAM8 protein from Saccharomyces cerevisiae. As a polyclonal IgG antibody, it contains multiple antibody clones that bind different epitopes across the NAM8 protein structure . This polyclonal nature increases detection sensitivity compared to monoclonal alternatives.

The antibody's ability to recognize the native protein conformation in applications like ELISA while also functioning in denaturing conditions (Western blotting) suggests that it recognizes both conformational and linear epitopes. This characteristic is beneficial for researchers conducting experiments under various conditions or using different experimental techniques.

What are the optimal storage and handling conditions for NAM8 Antibody?

For maximum stability and activity retention, NAM8 Antibody should be stored at either -20°C or -80°C immediately upon receipt. The antibody is supplied in liquid form in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody stability during freeze-thaw cycles.

To preserve antibody integrity and activity:

• Avoid repeated freeze-thaw cycles

• Aliquot the antibody into smaller volumes before freezing if multiple uses are anticipated

• Allow the antibody to equilibrate to room temperature before opening

• Handle using sterile technique to prevent contamination

• Return to appropriate storage temperature promptly after use

How can NAM8 Antibody be optimized for chromatin immunoprecipitation (ChIP) experiments?

While NAM8 Antibody is primarily validated for ELISA and Western blotting applications , researchers interested in ChIP applications should conduct preliminary optimization experiments. Since NAM8 functions in RNA processing, ChIP experiments may reveal valuable insights into its potential chromatin associations during transcription.

Optimization protocol for ChIP applications:

• Cross-validation: First confirm antibody specificity via Western blot in your specific yeast strain

• Cross-linking optimization: Test both formaldehyde concentrations (0.75%, 1%, and 1.5%) and incubation times (10, 15, and 20 minutes)

• Sonication parameters: Optimize to achieve chromatin fragments of 200-600bp

• Antibody titration: Test different concentrations (2μg, 5μg, and 10μg per reaction)

• Include appropriate controls:

  • Input chromatin (non-immunoprecipitated)

  • IgG negative control

  • Positive control targeting a known chromatin-associated protein

The Protein A/G purification method used for this antibody makes it compatible with standard ChIP protocols using protein A/G magnetic beads.

What experimental approaches can determine NAM8 binding partners in yeast?

NAM8 protein functions within complex RNA processing machinery. To investigate its binding partners, consider the following methodological approaches:

• Co-immunoprecipitation (Co-IP) with NAM8 Antibody:

  • Lyse yeast cells under non-denaturing conditions

  • Use NAM8 Antibody for immunoprecipitation

  • Analyze precipitated complexes by mass spectrometry or Western blotting

• Yeast Two-Hybrid Screening:

  • Use NAM8 as bait protein

  • Screen against a yeast cDNA library

  • Validate interactions with Co-IP using NAM8 Antibody

• Proximity-Dependent Biotin Identification (BioID):

  • Generate NAM8-BioID fusion construct

  • Express in yeast cells and activate biotinylation

  • Purify biotinylated proteins and analyze by mass spectrometry

  • Confirm interactions using NAM8 Antibody

Each approach offers complementary information about protein-protein interactions, and using NAM8 Antibody for validation ensures specificity in confirming potential binding partners.

How does NAM8 protein function compare across different yeast species?

NAM8 protein is highly conserved across Saccharomyces species, but with functional variations in other fungi. The NAM8 Antibody specificity for S. cerevisiae (strain ATCC 204508/S288c) must be considered when designing comparative studies.

Table 1: Cross-Reactivity Prediction of NAM8 Antibody Across Yeast Species

Researchers investigating NAM8 function across species should first validate antibody cross-reactivity in each species through Western blot analysis with appropriate controls before proceeding with comparative functional studies.

What controls should be included when using NAM8 Antibody in immunological applications?

Proper experimental controls are essential for ensuring result validity when using NAM8 Antibody:

Essential Controls for Western Blotting:

• Positive control: Wild-type S. cerevisiae extract containing NAM8 protein

• Negative control: NAM8 knockout yeast strain extract

• Loading control: Antibody against a constitutively expressed yeast protein (e.g., actin)

• Antibody specificity control: Pre-incubation of NAM8 Antibody with recombinant NAM8 protein to block specific binding

• Secondary antibody control: Sample incubated with secondary antibody only

Essential Controls for ELISA:

• Standard curve: Serial dilutions of recombinant NAM8 protein

• Blank wells: Buffer only (no antigen or antibody)

• Secondary antibody control: Wells without primary antibody

• Non-specific binding control: Irrelevant rabbit polyclonal IgG

These controls help distinguish specific signals from background noise and validate experiment reliability and reproducibility.

How can researchers troubleshoot weak or non-specific signals when using NAM8 Antibody?

When encountering issues with NAM8 Antibody performance, consider the following troubleshooting approaches:

For Weak Signals:

• Antibody concentration: Increase primary antibody concentration (try 1:500, 1:250, 1:100 dilutions)

• Incubation time: Extend primary antibody incubation (4°C overnight instead of 1-2 hours)

• Detection system: Switch to more sensitive detection method (e.g., ECL Plus, fluorescent secondary antibodies)

• Protein loading: Increase total protein amount loaded

• Blocking optimization: Test different blocking agents (BSA, milk, commercial blockers)

For Non-specific Signals:

• Washing stringency: Increase wash buffer stringency (add 0.1-0.5% Tween-20)

• Blocking time: Extend blocking step to 2 hours or overnight

• Antibody dilution: Prepare antibody in fresh blocking buffer

• Cross-adsorption: Pre-adsorb antibody with yeast lysate lacking NAM8

• Filter membrane: Pre-filter antibody solution through 0.22μm filter

Table 2: Systematic Optimization Matrix for NAM8 Antibody in Western Blotting

How should researchers interpret variations in NAM8 protein expression across different growth conditions?

NAM8 expression and function can vary significantly with growth conditions and stress responses in yeast. When analyzing results across different experimental conditions:

• Normalize properly: Always normalize NAM8 protein levels to appropriate housekeeping proteins that remain stable under your experimental conditions

• Consider post-translational modifications: NAM8 may undergo modifications affecting antibody recognition; examine potential band shifts in Western blots

• Evaluate subcellular localization: NAM8 distribution may change under different conditions; complement Western blot data with immunofluorescence studies

• Analyze time-course experiments: NAM8 expression can change dynamically; collect samples at multiple time points

• Correlate with functional data: Connect expression changes with functional readouts (e.g., mRNA splicing efficiency of NAM8-dependent transcripts)

Researchers should examine both absolute expression levels and relative changes compared to baseline conditions, interpreting data in the context of the experimental system's physiological state.

What bioinformatic approaches can enhance NAM8 protein research when combined with antibody-based detection?

Integrating experimental data from NAM8 Antibody studies with bioinformatic analyses creates powerful research strategies:

• Motif analysis: Identify RNA binding motifs in transcripts affected by NAM8 manipulation

  • Tools: MEME Suite, RSAT, RNApromo

• Structural prediction: Model NAM8 protein domains and interaction interfaces

  • Tools: AlphaFold, I-TASSER, PyMOL for visualization

• Pathway enrichment: Analyze transcripts and proteins affected by NAM8 perturbation

  • Tools: DAVID, STRING, KEGG Pathway analysis

• Evolutionary analysis: Compare NAM8 sequence and function across species

  • Tools: MEGA, PhyML, PAML

• Integrated network analysis: Combine protein-protein interaction data with transcriptomic and proteomic datasets

  • Tools: Cytoscape, NetworkAnalyst

These computational approaches transform descriptive antibody-based observations into mechanistic insights about NAM8 function in cellular processes.

How can researchers effectively validate NAM8 Antibody specificity in their specific experimental systems?

Thorough validation of NAM8 Antibody specificity is critical for result interpretation. A comprehensive validation approach includes:

• Genetic controls:

  • Compare wild-type strains with NAM8 deletion strains

  • Use strains with epitope-tagged NAM8 for parallel detection

• Molecular weight verification:

  • Confirm that detected bands match the predicted molecular weight (52.6 kDa for NAM8)

  • Look for expected shifts with tagged versions of the protein

• Competition assays:

  • Pre-incubate antibody with purified recombinant NAM8 protein before immunodetection

  • Signal should decrease proportionally to competing protein concentration

• Orthogonal detection methods:

  • Compare results using alternative NAM8 detection methods (e.g., mass spectrometry)

  • Detect NAM8 using antibodies recognizing different epitopes

• Immunodepletion:

  • Perform sequential immunoprecipitations to confirm complete depletion

  • Validate by Western blotting of supernatant and precipitate fractions

Validation should be performed in the specific yeast strain and under the experimental conditions relevant to the study.