SPBC660.17c Antibody

What is SPBC660.17c and what is known about its function?

SPBC660.17c is a gene from Schizosaccharomyces pombe (fission yeast) that encodes an uncharacterized membrane protein C660.17c. It is classified as a "sequence orphan," meaning it doesn't have known homologs in other organisms . The protein consists of 172 amino acids with the sequence: MEQLRKRVVRFTNNDDDDFEPVFLNEQDQDAFVEQLRLTNNRDNRMFSIIFSFLYLLLIV PLFLYPEYWAFKLVELLSLFYCAYVMYFLPLEVGLFNPKTPNKWKFLFILNIGVTALITV LGWSQHTSFFYAFLNIRTLVCGITIFTEIARYSMYHSTLSVEKLDEMRFAHM . The transmembrane domains in the sequence suggest it functions as a membrane protein, though its specific biological role remains to be elucidated.

What type of antibody is available for SPBC660.17c detection?

The SPBC660.17c antibody (CSB-PA528441XA01SXV-0.2) is a rabbit polyclonal antibody that specifically recognizes the SPBC660.17c protein from Schizosaccharomyces pombe (strain 972 / ATCC 24843) . The antibody is raised against recombinant SPBC660.17c protein and is purified by antigen affinity chromatography . This format allows recognition of multiple epitopes on the target protein, potentially increasing detection sensitivity.

How is the SPBC660.17c antibody typically produced?

Based on similar yeast protein antibody production methods, SPBC660.17c antibody is likely produced by:

• Cloning the SPBC660.17c gene from S. pombe genomic DNA

• Expressing it as a recombinant protein in E. coli (similar to the method described for Rhb1 antibody production)

• Purifying the recombinant protein using affinity chromatography

• Immunizing rabbits with the purified protein to generate polyclonal antibodies

• Harvesting and purifying the antibodies from rabbit serum using antigen affinity methods

What experimental applications are validated for SPBC660.17c antibody?

The SPBC660.17c antibody is validated for:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blot (WB)

These applications allow detection and relative quantification of SPBC660.17c protein in various experimental contexts. The antibody components typically include 200μg of antigen (for positive control) and 1ml of pre-immune serum (for negative control), facilitating proper experimental design .

What is the recommended protocol for Western blot using SPBC660.17c antibody?

While specific protocols may vary, a general Western blot procedure would include:

• Sample preparation:

  • Lyse S. pombe cells using detergent-based buffers (necessary for membrane proteins)

  • Include protease inhibitors to prevent degradation

  • Determine protein concentration (Bradford or BCA assay)

• SDS-PAGE and transfer:

  • Load 20-50μg of total protein per lane

  • Separate proteins by SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane

• Immunoblotting:

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

  • Incubate with SPBC660.17c antibody (optimal dilution to be determined experimentally, typically 1:500-1:2000)

  • Wash 3-5 times with TBST

  • Incubate with appropriate secondary antibody (anti-rabbit IgG-HRP)

  • Develop using chemiluminescence detection

• Controls:

  • Include positive control (provided antigen)

  • Include negative control (pre-immune serum)

  • Include lysate from SPBC660.17c deletion strain when possible

How should the SPBC660.17c antibody be stored and handled?

For optimal performance and longevity:

• Store at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• For short-term storage (up to one week), keep working aliquots at 4°C

• Use a manual defrost freezer (similar to recommendations for other antibodies)

• Handle with clean gloves and pipettes to prevent contamination

• Avoid vortexing to prevent antibody denaturation

How can I validate the specificity of the SPBC660.17c antibody?

To validate antibody specificity:

• Perform Western blot analysis using:

  • Wild-type S. pombe lysate (should show band at expected MW)

  • SPBC660.17c deletion strain lysate (should show no band)

  • Recombinant SPBC660.17c protein as positive control

• Conduct pre-absorption test:

  • Pre-incubate antibody with recombinant SPBC660.17c protein

  • Use pre-absorbed antibody in Western blot

  • Signal should be significantly reduced or eliminated

• Compare results with pre-immune serum:

  • Perform parallel Western blot with pre-immune serum

  • No specific binding should be observed

What positive and negative controls should be used with SPBC660.17c antibody?

For rigorous experimental design, include:

Positive controls:

• Recombinant SPBC660.17c protein (provided with antibody, 200μg)

• Wild-type S. pombe lysate expressing SPBC660.17c

Negative controls:

• Pre-immune serum (provided with antibody, 1ml)

• S. pombe SPBC660.17c deletion strain lysate (if available)

• Primary antibody omission control

• Unrelated yeast species lysates (e.g., S. cerevisiae)

How do I determine the optimal antibody dilution for my experiment?

To determine optimal antibody concentration:

• Perform a dilution series experiment:

  • Test multiple dilutions (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Use consistent sample amounts and detection methods

  • Evaluate signal-to-noise ratio for each dilution

• Consider titration matrix:

  Antibody Dilution	Signal Strength	Background	Signal-to-Noise Ratio
  1:500	Strong	High	Medium
  1:1000	Strong	Medium	High
  1:2000	Medium	Low	High
  1:5000	Weak	Low	Low

• Select the dilution with optimal signal-to-noise ratio, typically where specific signal is clear with minimal background

What are common issues when using SPBC660.17c antibody and how can they be resolved?

Common problems and solutions include:

• Weak or no signal:

  • Increase antibody concentration

  • Extend incubation time

  • Verify protein expression levels

  • Use more sensitive detection methods

  • Check antibody storage conditions

• High background:

  • Increase blocking time/concentration

  • Use more stringent washing conditions

  • Reduce antibody concentration

  • Try different blocking agents (milk vs. BSA)

  • Prepare fresh buffers

• Multiple bands:

  • Verify with positive/negative controls

  • Include protease inhibitors during sample preparation

  • Optimize sample preparation for membrane proteins

  • Consider post-translational modifications or protein complexes

  • Test specificity with pre-absorption

How can SPBC660.17c antibody be used for membrane protein localization studies?

While primarily validated for ELISA and Western blot, adaptation for localization studies requires:

• Optimization of fixation methods:

  • Test different fixatives (paraformaldehyde, methanol, acetone)

  • Optimize fixation time and temperature

  • Ensure adequate permeabilization for membrane proteins

• Immunofluorescence protocol development:

  • Test higher antibody concentrations (typically 2-5× more than Western blot)

  • Extend primary antibody incubation (overnight at 4°C)

  • Optimize blocking to reduce non-specific binding

  • Use appropriate fluorescent secondary antibodies

• Controls for localization studies:

  • Include SPBC660.17c deletion strain

  • Use specific membrane markers for co-localization

  • Perform Z-stack imaging to visualize membrane distribution

Can the SPBC660.17c antibody be used for co-immunoprecipitation to identify interaction partners?

Co-immunoprecipitation (Co-IP) with SPBC660.17c antibody requires:

• Crosslinking optimization:

  • Test various crosslinkers (DSP, formaldehyde) at different concentrations

  • Optimize crosslinking time for membrane protein complexes

• Membrane protein solubilization:

  • Test different detergents (digitonin, CHAPS, NP-40)

  • Adjust salt and detergent concentrations to maintain interactions

  • Use gentle lysis methods to preserve protein complexes

• Immunoprecipitation protocol:

  • Conjugate SPBC660.17c antibody to protein A/G beads or magnetic beads

  • Pre-clear lysates to reduce non-specific binding

  • Optimize antibody amount and incubation conditions

  • Include appropriate controls (pre-immune serum, irrelevant antibody)

• Analysis of co-precipitated proteins:

  • Use mass spectrometry for unbiased partner identification

  • Confirm interactions with reciprocal Co-IP when possible

How can researchers utilize the SPBC660.17c antibody to study protein expression under stress conditions?

To investigate stress responses:

• Experimental design:

  • Subject S. pombe cultures to various stresses (oxidative, heat, nutrient limitation)

  • Collect samples at multiple time points

  • Extract proteins using optimized protocols for membrane proteins

• Quantitative analysis:

  • Use Western blot with SPBC660.17c antibody

  • Include loading controls (e.g., α-tubulin antibody)

  • Quantify band intensities using densitometry

  • Normalize to loading controls

• Correlation with phenotypic data:

  • Compare protein expression patterns with growth curves

  • Analyze in conjunction with transcriptomic data

  • Test SPBC660.17c deletion strain responses to the same stresses

How does SPBC660.17c compare to other membrane proteins in S. pombe?

Comparative analysis involves:

• Sequence alignment:

  • Analyze transmembrane domains with other S. pombe membrane proteins

  • Compare with predicted membrane topologies

  • Identify potential functional domains

• Expression pattern comparison:

  • Use SPBC660.17c antibody alongside antibodies for known membrane proteins

  • Compare expression levels across growth conditions

  • Analyze co-regulation patterns

• Phylogenetic analysis:

  • Although classified as a sequence orphan, compare with distant homologs

  • Search for structural similarities with characterized membrane proteins

How can SPBC660.17c antibody be used in genomewide screens for drug sensitivity?

In antifungal drug research contexts:

• Protein expression analysis:

  • Treat wild-type and SPBC660.17c deletion strains with antifungal compounds

  • Use Western blot to monitor expression changes of SPBC660.17c and other proteins

  • Correlate expression changes with growth inhibition patterns

• Integration with genomic data:

  • Compare protein expression data with results from deletion library screens

  • Analyze potential involvement in drug resistance mechanisms

  • Investigate membrane protein complexes affected by antifungal treatments

• Experimental design table:

  Condition	Strains	Analysis Method	Expected Outcome
  Control	WT, ΔSPBC660.17c	Western blot	Baseline expression
  Drug A	WT, ΔSPBC660.17c	Western blot	Expression changes
  Drug B	WT, ΔSPBC660.17c	Western blot	Expression changes
  Combined drugs	WT, ΔSPBC660.17c	Western blot	Synergistic effects

What methods can be used to investigate post-translational modifications of SPBC660.17c?

Advanced PTM analysis approaches:

• Western blot analysis:

  • Look for mobility shifts indicating modifications

  • Use phosphatase treatment to identify phosphorylation

  • Compare different growth conditions for dynamic modifications

• Mass spectrometry:

  • Immunoprecipitate SPBC660.17c using the antibody

  • Perform tryptic digestion and MS/MS analysis

  • Map identified modifications to protein sequence

• Modification-specific antibodies:

  • Use in parallel with SPBC660.17c antibody

  • Compare detection patterns under different conditions

  • Correlate modifications with protein localization and function