MOUSE ANTI LISTERIA MONOCYTOGENES Antibody (0.2mg)

Catalog#: OASA03452

• Size: 0.2mg

• Description of Target: MOUSE ANTI LISTERIA MONOCYTOGENES
• Alias Symbols: N/A
• Host: Mouse
• Specificity: LISTERIA MONOCYTOGENES
• Application: ELISA, WB
• Clone: LZF7 (BGN/0884/67)
• Immunogen: Listeria monocytogenes, outer membrane fraction.
• Product Format: Purified IgG - liquid
• Isotype: IgG2a
• Applications Info: ELISA : This product is suitable for use as a detection antibody in a sandwich ELISA, with OASA03453 as the coating antibody.
  
  Western Blot: This product detects a band of approximately 23kDa in reduced blots of cell fragments.
  Preparation: Purified IgG prepared by affinity chromatography on Protein G.
  
  Preservative Stabilisers: 0.09% Sodium Azide (NaN₃)
  Approx Protein Conc: IgG concentration 1.0 mg/ml
  
  Buffer Solutions: Phosphate buffered saline, pH 7.2
• Reconstitution and Storage: Store at +4^oC or at -20^oC if preferred.
  Storage in frost-free freezers is not recommended.
  This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.

Product Protocol: MOUSE ANTI LISTERIA MONOCYTOGENES antibody used to evaluate priming of protective anti-listeria monocytogenes memory cd8+ t cells requires a functional seca2 secretion system (OASA03452)

Specificity: LISTERIA MONOCYTOGENES
Product Page: MOUSE ANTI LISTERIA MONOCYTOGENES antibody (OASA03452)
Antibody Pair Available: OASA03453 Capture OASA03452 Detection
Experiment Type: Western blotting

Protocol:
Western blotting: (i) L. monocytogenes secretome: Proteins from O/N culture supernatants of NBS- and Del-SecA2 mutants and wt bacteria were precipitated by treatment with trichloroacetic acid (TCA) at a final concentration of 6% on ice for 30 min. Precipitates were washed with cold acetone, dried, and suspended in SDS-PAGE loading buffer (50 mM Tris-Cl [pH 6.8], 2% SDS, 10% glycerol, 0.1% bromophenol blue) containing 5% dimethyl sulfoxide (DMSO). Samples were boiled for 5 min, run on an SDS–10% polyacrylamide gel, transferred to nitrocellulose, and probed with Listeria rabbit antiserum (BD Difco). Goat anti-rabbit IgG-peroxidase (Beckman Coulter) was used as a secondary probe, and bound antiserum was revealed using the Super Signal West Femto kit (Pierce).

(ii) Expression of wt and NBS mutant SecA2: Bacteria were grown in brain heart broth medium (Fluka) to exponential phase, and ∼1010 bacteria were pelleted, washed in phosphate-buffered saline (PBS)–1 mM phenylmethylsulfonyl fluoride (PMSF), and incubated in 40 mM Tris-HCl–150 mM NaCl containing 10 mg/ml chicken egg white lysozyme M (Sigma) at 37°C for 90 min in the presence of a cocktail of protease inhibitors (Thermo Scientific). The bacterial suspension was then sonicated (Misonix Ultrasonic Processors) with 1-min pulses for 20 min, pelleted, resuspended in PBS–1% SDS, and incubated for 30 min at 37°C. Approximately 3.3 × 108 bacteria from the initial culture were loaded in SDS-PAGE sample buffer (62.5 mM Tris [pH 6.8], 2% SDS, 10% glycerol, 0.1 M dithiothreitol [DTT], 0.01% bromophenol blue) and separated on an 8% SDS-polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane, probed with a horseradish peroxidase (HRP)-coupled anti-FLAG monoclonal antibody (MAb) (clone M2; Sigma), and further revealed (Chemiglow West substrate) using the FluorChem FC2 imaging system (Alpha Innotech). Quantification was performed with AlphaView software.

Summary:
1. While some L. monocytogenes-derived proteins exhibited equal intensities in the supernatants from all bacteria, many other proteins detected in the culture supernatant of wt bacteria were reduced in NBS- and in Del-SecA2 bacteria to comparable extents. Next, Massilva et al. tested whether the NBS-SecA2 mutant of L. monocytogenes was also impaired in its virulence in vivo. They found that the mutants exhibit comparable LD50s in mice (107 bacteria) (data not shown), a result in agreement with published reports on Del-SecA2 L. monocytogenes by them and others. 2. Recombinant or wt (control) bacteria were exponentially grown, and the whole bacterial pellet was extracted, Western blotted, and revealed using an anti-FLAG specific MAb. The results revealed that wt, NBS-I-mutated, and NBS-I- and -II-mutated SecA2 proteins were detected at the expected molecular mass (∼85 kDa) (not shown) and expressed at equivalent levels in all independently selected recombinant bacterial clones. Collectively these experiments therefore suggested that mutating the NBS inside the SecA2 protein affects primarily its ATPase function but not its expression levels inside the bacteria. Of note, mutating the NBS-I Walker site seems to be sufficient for the observed phenotype.

References:
1: Rahmoun M, Gros M, Campisi L, Bassand D, Lazzari A, Massiera C, Narni-Mancinelli E, Gounon P, Lauvau G. Priming of protective anti-Listeria monocytogenes memory CD8+ T cells requires a functional SecA2 secretion system. Infect Immun. 2011 Jun;79(6):2396-403. Epub 2011 Mar 14. PubMed PMID: 21402759; PubMed Central PMCID: PMC3125821.