User:Vijay/Protocol/saureus-fractionation

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Contents

Overview

I do Staphylococcus aureus cell fractionation to separate proteins from different locations, like cytoplasmic, membrane, cell wall and extracellular. The complete procedure is used mostly during localization studies. The procedure could also be broken at specific point, if you are interested in only any one of the fractions.

Materials

  • Digestion buffer (Tris HCL)
  • Raffinose (dissolves easily at 50-55oC water bath)
  • PBS
  • Lysostaphin (stock 10mg/ml)
  • TSB (500ml in 2 liter conical flask)
  • Protease inhibitor (1tablet in 1ml ddwater)
  • Centrifuge tubes (Beckman)
  • Rotor (Type 70 Ti)
  • Centricon

Procedure

  • Day ONE
  1. Streak out culture from stock
  • Day TWO
  1. Inoculate in to 5ml TSB
  2. Inoculate 200ul of ON culture in to 500ml TSB, 250rpm
  1. 50ml in 4 Falcon tubes (50x4=200ml)
  2. Spun at 8700rpm, 15min, 4oC
  3. Collect 100ml of supernatant (filter using .45um filter)
  4. Wash with Ice cold PBS (5ml), by vortexing and pool in to one tube
  5. Spun at 8700rpm, 5min, 4oC
  6. Suspend in 5ml PBS, OD at 560nm (100ul +900ul) (usually around OD 25)
  7. Spun at 8700rpm, 5min, 4oC
  • CELL WALL FRACTION
  1. Resuspend in 5ml Raffinose buffer (raffinose + digesion buffer), add 500ul protease inhibitor, add 200ul of lysostaphin, incubate at 37oC for 1hr
  2. spun at 8000rpm, 20-40min, 4oC to separate cell wall fraction
  • CYTOPLASMIC FRACTION
  1. Wash protoplast with raffinose buffer
  2. Resuspend protoplast using cold digestion (lysis) buffer
  3. Protoplast lysed by using FAST PREP tubes, at 4.5 speed and 40s setup
  4. Lysates pooled, centrifuged at 3000g, 4oC, 2-30min to remove cell debris
  5. Supernatant transferred to Beckman centrifuge tubes
  6. Balance water (both tubes measured for equal weight)
  7. 40,000g or 23,400rpm for membrane fraction, Acel and Decel - both 7, Temp 4oC, Time 1hr
  8. Vacuum (has to come down atleast to 50, takes about 30-40minutes)
  9. Start centrifuging
  10. Remove supernatant (Cytoplasmic fraction)
  • MEMBRANE FRACTION
  1. Wash pellet with ice cold digestion (lysis) buffer
  2. Resuspend in 5ml lysis buffer
  • EXTRACELLULAR FRACTION
  1. 3ml of supernatant concentrated using CENTRICON, overnight at 4oC, Pooled together

COULD BE DIRECTLY USED IN SDS-PAGE without concentrating using TCA

Reference

  • Robert Downer et al., JBC,2003, Vol 277(1).243-250. The Elastin-binding Protein of Staphylococcus aureus (EbpS)...
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