绿脓杆菌的细胞分级分离

Esteban  Paredes-Osses; Kim R.  Hardie

doi:10.21769/BioProtoc.922

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Peer-reviewed

Cell Fractionation of Pseudomonas aeruginosa

绿脓杆菌的细胞分级分离

Esteban Paredes-Osses email

Kim R. Hardie email

发布: 2013年10月05日第3卷第19期 DOI: 10.21769/BioProtoc.922 浏览次数: 12965

评审: Fanglian He

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Aug 2012

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Abstract

Pseudomonas aeruginosa is a Gram negative bacterium. Separating the cell envelope compartments enables proteins to be localized to confirm where in the cell they function. Cell fractionation can also provide a first step in a protein purification strategy (Williams et al., 1998). This protocol has been designed to obtain the different fractions of P. aeruginosa, namely the inner membrane, outer membrane, cytoplasmic and periplasmic compartments. Specific detection of the arginine specific autotransporter (AaaA) (Luckett et al., 2012) in the outer membrane of P. aeruginosa has been performed using this protocol.

Materials and Reagents

P. aeruginosa strain (In this experiment, AaaA deficient mutant strains are used). The strain was bearing the pME6032 shuttle expression vector (Heeb et al., 2002). This vector is IPTG-inducible and has very good stability in Pseudomonas.
Polyclonal antibody anti-AaaA (rabbit) (not commercial available) (Luckett et al., 2012)
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat (Sigma-Aldrich, catalog number: A6154 )
Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I5502 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Luria Bertani (LB) (Difco, catalog number: 244620 )
Autoclaved sterilized Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417 )
Sucrose (Sigma-Aldrich, catalog number: S7903 )
Sodium Lauryl Sarcoscine (SLS) (Sigma-Aldrich, catalog number: L5125 )
Magnesium Sulphate (Sigma-Aldrich, catalog number: 63139 )
MgCl₂ (Sigma-Aldrich, catalog number: M2393 )
DNAase I (Sigma-Aldrich, catalog number: D4527 )
RNAase A (Sigma-Aldrich, catalog number: R5503 )
100% Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
Acetone (Sigma-Aldrich, catalog number: 179124 )
NaOH (Sigma-Aldrich, catalog number: 221465 )
Tris(hydroxymethyl) aminomethane (Sigma-Aldrich, catalog number: 252859 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
Sodium Dodecyl Sulphate (SDS) (Sigma-Aldrich, catalog number: L4509 )
Bromophenol Blue (Sigma-Aldrich, catalog number: B0126 )
Glycerol (Sigma-Aldrich, catalog number: G8773 )
4x Loading buffer (see Recipes)

Equipment

French Pressure cell (Manufactured by Amicon and supplied by Thermo Fisher Scientific)
50 ml Falcon tube (BD Biosciences, Falcon^®, catalog number: 352070 ) (Supplied by Scientific Laboratory Supplies)
Eppendorf^® Safe-Lock micro test tubes (Eppendorf, catalog number: 0030121023 ) (Supplied by Scientific Laboratory Supplies)
Microfuge, larger volume centrifuge and high speed centrifuge (Beckman Coulter, model: Allegra X-22R Centrifuge )
1 ml pipettman
W380 sonicator (Ultrasonics)

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Paredes-Osses, E. and Hardie, K. R. (2013). Cell Fractionation of Pseudomonas aeruginosa. Bio-protocol 3(19): e922. DOI: 10.21769/BioProtoc.922.
Luckett, J. C., Darch, O., Watters, C., Abuoun, M., Wright, V., Paredes-Osses, E., Ward, J., Goto, H., Heeb, S., Pommier, S., Rumbaugh, K. P., Camara, M. and Hardie, K. R. (2012). A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity. PLoS Pathog 8(8): e1002854.