SPBC359.01 Antibody

What is SPBC359.01 and what cellular functions does it perform?

SPBC359.01 is an uncharacterized amino acid permease found in Schizosaccharomyces pombe (strain 972/24843), commonly known as fission yeast. Based on sequence analysis, it contains PROSITE amino acid permease signature motifs and is predicted to have 12 transmembrane domains with an intracellular 87 amino acid N-terminal tail and 47 amino acid C-terminal tail . This structure is strikingly similar to that of Saccharomyces cerevisiae Can1, despite sharing only 14.8% amino acid sequence identity .

Functionally, SPBC359.01 appears to be involved in amino acid transport across cellular membranes. Studies have shown that disruption of SPBC359.01 results in defects in arginine and lysine uptake, similar to those observed in Δtsc2 mutants . This suggests that SPBC359.01 is regulated by the Tsc/Rheb signaling pathway, which controls basic amino acid uptake in fission yeast.

How are SPBC359.01 antibodies typically validated for research use?

SPBC359.01 antibodies are typically validated through several complementary approaches:

• Western blotting: Whole cell extracts are prepared by resuspending cells in lysis buffer (20 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1.6% SDS, 6 M urea) containing protease inhibitor mixture. After SDS-PAGE, proteins are transferred to a nitrocellulose membrane and probed with anti-SPBC359.01 antibodies .

• ELISA: This technique confirms antibody specificity by coating plates with recombinant SPBC359.01 protein and detecting binding with the antibody of interest. This validation method is commonly used for polyclonal antibodies against SPBC359.01 .

• Immunofluorescence: For subcellular localization studies, the antibody can be tested on wild-type versus SPBC359.01 knockout cells to confirm specificity of staining patterns.

• Peptide competition assays: Pre-incubating the antibody with the immunogenic peptide should abolish specific signals in any of the above assays.

What are the recommended experimental conditions for using SPBC359.01 antibodies in Western blotting?

For optimal Western blot results with SPBC359.01 antibodies, the following experimental conditions are recommended:

• Sample preparation: Harvest 50 ml of S. pombe cells grown to OD600 1.0−2.0. Resuspend in 250 μl of lysis buffer (20 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1.6% SDS, 6 M urea) containing protease inhibitor mixture (1 mM PMSF and 1× protease inhibitor cocktail) .

• Protein separation: Load 50 μg of protein sample on a 12.5% SDS-polyacrylamide gel and run electrophoresis in SDS-tris-glycine buffer .

• Transfer conditions: Transfer proteins to a nitrocellulose membrane using standard Western blotting protocols.

• Blocking: Block the membrane with 1-5% BSA in PBS or PBST (PBS with 0.05% Tween-20).

• Primary antibody incubation: Dilute anti-SPBC359.01 antibody 1:1000 to 1:10,000 in blocking buffer and incubate overnight at 4°C or for 1-2 hours at room temperature.

• Secondary antibody: Use an appropriate HRP-conjugated secondary antibody at 1:4,000 to 1:10,000 dilution for 1 hour at room temperature .

• Detection: Develop using ECL reagent and image using an appropriate imaging system.

Note: Always include appropriate controls such as a loading control (anti-tubulin or anti-PCNA) and, if possible, samples from SPBC359.01 knockout strains to confirm antibody specificity .

How is SPBC359.01 expression typically measured in cells?

SPBC359.01 expression can be measured at both the mRNA and protein levels using the following techniques:

mRNA level measurement:

• Northern blot analysis: Ten micrograms of total RNA is run on a 4% formaldehyde gel and transferred to a nylon membrane. Probes for SPBC359.01 and control genes (e.g., tub1+) can be PCR-amplified from cDNA, labeled with [α32P] dATP, and used for hybridization in Quickhyb buffer .

• Real-time RT-PCR: Approximately 1 μg of total cellular RNA is reverse transcribed using a high-capacity reverse transcription kit. The resulting cDNA is then subjected to real-time PCR with SYBR green PCR master mix and transcript-specific primers. Results are typically normalized to an internal control like GAPDH (Spbc32f12.11) .

• RNA-Seq: This approach provides comprehensive analysis of transcriptomes and can quantify SPBC359.01 expression relative to all other genes.

Protein level measurement:

• Western blot analysis: As described in the Western blotting protocol above, using anti-SPBC359.01 antibodies and appropriate controls .

• Mass spectrometry: For more precise quantification, targeted proteomics approaches like selected reaction monitoring (SRM) can be employed.

How can SPBC359.01 antibodies be used to investigate protein-protein interactions in fission yeast?

SPBC359.01 antibodies can be valuable tools for investigating protein-protein interactions through several sophisticated approaches:

• Co-immunoprecipitation (Co-IP):

  • Prepare cell lysates in a non-denaturing buffer to preserve protein-protein interactions

  • Pre-clear the lysate with Protein A/G beads

  • Incubate with anti-SPBC359.01 antibody overnight at 4°C

  • Pull down with fresh Protein A/G beads

  • Analyze interacting partners by SDS-PAGE followed by Western blotting or mass spectrometry

• Proximity-dependent labeling combined with immunoprecipitation:

  • Express SPBC359.01 fused to a proximity labeling enzyme (BioID or APEX2)

  • After biotin labeling, use anti-SPBC359.01 antibodies to confirm proper expression and localization

  • Purify biotinylated proteins and identify by mass spectrometry

• Fluorescence microscopy with co-localization studies:

  • Perform immunofluorescence using anti-SPBC359.01 antibodies along with antibodies against potential interacting partners

  • Analyze co-localization patterns through confocal microscopy

• In situ Proximity Ligation Assay (PLA):

  • This method can detect protein-protein interactions directly in fixed cells

  • Use anti-SPBC359.01 antibody together with antibodies against suspected interaction partners

  • Proximity of the two target proteins (<40 nm) will generate a fluorescent signal

When investigating SPBC359.01 interactions, it's particularly relevant to examine relationships with components of the Tsc/Rheb signaling pathway, which has been shown to regulate amino acid permeases in fission yeast .

What approaches can be used to determine SPBC359.01 subcellular localization and trafficking?

Determining the subcellular localization and trafficking of SPBC359.01 requires combining antibody-based techniques with other cell biology approaches:

• Immunofluorescence microscopy:

  • Fix S. pombe cells with 3.7% formaldehyde

  • Permeabilize cell walls with zymolyase treatment

  • Incubate with anti-SPBC359.01 primary antibody and fluorophore-conjugated secondary antibody

  • Co-stain with markers for various organelles (plasma membrane, endosomes, Golgi, etc.)

• Subcellular fractionation with Western blotting:

  • Prepare cell extracts and separate cellular components through differential centrifugation

  • Analyze fractions by Western blotting with anti-SPBC359.01 antibody

  • Compare distribution with known compartment markers

• Live cell imaging with GFP-tagged SPBC359.01:

  • The C-terminus of SPBC359.01 can be tagged with GFP using PCR-based methods and the hphMX cassette

  • Verify proper expression and functionality using anti-SPBC359.01 antibodies

  • Track protein movement in response to environmental stimuli

• Detergent resistance analysis:

  • Membrane proteins can be analyzed for lipid raft association using TX-100 extraction

  • After treatment, load extracts onto Optiprep gradients and centrifuge at 200,000 g

  • Analyze fractions by Western blotting with anti-SPBC359.01 antibody

Research has shown that similar amino acid permeases may be regulated by endocytosis and trafficking in response to nutrient availability. Using anti-SPBC359.01 antibodies can help determine if this protein follows similar regulatory patterns.

How can researchers investigate the functional relationship between SPBC359.01 and ABC3 (SPBC359.05) transporters?

The genomic proximity and potential functional relationship between SPBC359.01 (amino acid permease) and ABC3/SPBC359.05 (ATP-binding cassette transporter) suggests possible coordinated regulation or functional interaction. To investigate this relationship:

• Comparative expression analysis:

  • Perform Northern blot or RT-PCR analysis for both genes under various conditions

  • Use antibodies against both proteins to examine if their expression is coordinated

  • Design experiments using riboprobes specific to SPBC359.01 and ABC3 as described in the literature

• Double knockout studies:

  • Generate single and double knockout strains (Δspbc359.01, Δabc3, and Δspbc359.01Δabc3)

  • Compare phenotypes using growth assays, nutrient uptake measurements, and stress response tests

  • Use antibodies to confirm absence of protein expression in respective knockout strains

• Transport assays:

  • Measure uptake of radiolabeled substrates ([3H]arginine, [3H]lysine) in wild-type and mutant strains

  • Compare transport kinetics between single and double mutants

  • Use protocol similar to that described for arginine uptake measurement :

    • Grow cells to mid-log phase (OD600 = 0.5–1.0)

    • Resuspend in uptake buffer

    • Measure incorporation of radiolabeled amino acids over time

• Iron and amino acid stress response:

  • Since ABC3 is an iron-regulated vacuolar ABC-type transporter , investigate if SPBC359.01 also responds to iron availability

  • Test both proteins' expression and localization under iron-depleted and iron-replete conditions

  • Use anti-SPBC359.01 and anti-ABC3 antibodies to track protein levels in these conditions

What methodological approaches should be used to study SPBC359.01 in membrane protein complexes?

Membrane protein complexes are challenging to study due to their hydrophobic nature and the need to maintain native interactions. For SPBC359.01, consider these specialized approaches:

• Blue native PAGE (BN-PAGE):

  • Solubilize membranes using mild detergents (digitonin, DDM, or CHAPS)

  • Separate native complexes by BN-PAGE

  • Perform Western blotting with anti-SPBC359.01 antibody

  • For second dimension, cut lanes from BN-PAGE and run on SDS-PAGE to separate complex components

• Crosslinking mass spectrometry (XL-MS):

  • Treat intact cells or isolated membranes with membrane-permeable crosslinkers

  • Immunoprecipitate SPBC359.01 using specific antibodies

  • Analyze crosslinked peptides by mass spectrometry to identify interacting partners

• GFP-Trap immunoprecipitation of tagged SPBC359.01:

  • Express SPBC359.01-GFP in S. pombe

  • Use GFP-Trap beads for efficient one-step immunoprecipitation

  • Confirm results using anti-SPBC359.01 antibodies in parallel experiments

  • Identify co-purifying proteins by mass spectrometry

• Sucrose gradient ultracentrifugation:

  • Solubilize membranes with appropriate detergents

  • Separate complexes by size on 10-40% sucrose gradients

  • Collect fractions and analyze by Western blotting with anti-SPBC359.01 antibody

  • Compare distribution with known membrane complex markers

• Lipidomics analysis of SPBC359.01-containing membranes:

  • Immunoprecipitate SPBC359.01 under native conditions

  • Extract and analyze associated lipids by mass spectrometry

  • Compare lipid profiles between wild-type and mutant proteins

When studying membrane proteins like SPBC359.01, it's critical to validate antibody specificity in the context of detergent-solubilized samples, as epitope accessibility may change in different solubilization conditions.

How can SPBC359.01 antibodies be used to investigate the TSC/Rheb signaling pathway's regulation of amino acid transport?

The TSC/Rheb signaling pathway plays a crucial role in regulating basic amino acid uptake in S. pombe. SPBC359.01 antibodies can be instrumental in dissecting this regulatory mechanism:

• Signaling pathway activation analysis:

  • Treat cells with rapamycin or other TOR pathway inhibitors

  • Monitor SPBC359.01 protein levels by Western blotting

  • Track changes in subcellular localization using immunofluorescence

  • Compare with known TSC/Rheb pathway components

• Phosphorylation state analysis:

  • Immunoprecipitate SPBC359.01 using specific antibodies

  • Analyze phosphorylation status by:

    • Phospho-specific antibodies (if available)

    • Western blotting with anti-phosphoserine/threonine antibodies

    • Mass spectrometry to identify phosphorylation sites

  • Compare phosphorylation patterns in wild-type vs. Δtsc2 mutants

• Regulated protein degradation:

  • Treat cells with cycloheximide to inhibit protein synthesis

  • Monitor SPBC359.01 stability over time by Western blotting

  • Compare degradation rates in wild-type vs. TSC/Rheb pathway mutants

  • Investigate involvement of ubiquitin-proteasome system

• Nutrient response studies:

  • Starve cells of specific amino acids and monitor SPBC359.01 expression

  • Use Northern blot analysis for mRNA levels as described in the literature

  • Use anti-SPBC359.01 antibodies to track protein levels and localization

  • Compare with response patterns of other amino acid permeases

Research has shown that disruption of SPBC359.11 results in defective arginine and lysine uptake similar to Δtsc2 mutants , suggesting a functional relationship between SPBC359.01 and the TSC/Rheb pathway. Using antibodies against both SPBC359.01 and TSC pathway components can help elucidate the molecular mechanisms of this regulation.

What are the recommended approaches for troubleshooting non-specific binding when using SPBC359.01 antibodies?

Non-specific binding is a common challenge when working with antibodies against membrane proteins like SPBC359.01. Here are systematic troubleshooting approaches:

• Validation with knockout controls:

  • Always include samples from SPBC359.01 knockout strains

  • Any signal in knockout samples indicates non-specific binding

  • Compare signal patterns between wild-type and knockout samples

• Optimization of blocking conditions:

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

  • Optimize blocking time and temperature

  • Consider adding 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform titration experiments with increasing antibody dilutions

  • Find the optimal concentration that maximizes specific signal while minimizing background

  • Consider longer incubation times with more dilute antibody solutions

• Sample preparation modifications:

  • For membrane proteins, test different detergents for cell lysis

  • Compare denaturing (SDS) vs. non-denaturing (digitonin, DDM) conditions

  • Evaluate whether heat denaturation affects epitope recognition

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Run parallel Western blots with competed and non-competed antibody

  • Specific signals should disappear in the competed sample

• Alternative detection methods:

  • Try fluorescent secondary antibodies instead of HRP-based detection

  • Consider direct labeling of primary antibodies to eliminate secondary antibody issues

  • Use signal amplification systems for weak but specific signals

When troubleshooting SPBC359.01 antibodies specifically, remember that membrane proteins like amino acid permeases can form aggregates during sample preparation, potentially leading to high molecular weight bands or smears on Western blots.