DGK Membrane Preparation

Reagents : Bacterial strain E. coli N4830/pJW10 LB amp media 50 µg/ml ampicillin High salt buffer for 1 L 50 mM KH2PO4 6.8 g 150 mM KCl 11.18 g 50 mM sodium pyrophosphate (Na4P2O7-10H2O) 22.3 g 5 mM Na2 EDTA 1.86 g 5 mM b-mercaptoethanol 390 µl TNE buffer for 1 L 25 mM Tris 3.03 g 100 mM NaCl 5.84 g 1 mM Na2 EDTA (pH 8.0) 0.37 g 5 mM b-mercaptoethanol 390 µl TEM buffer for 1 L 25 mM Tris 3.03 g 1 mM Na2 EDTA (pH 8.0) 0.37 g 5 mM b-mercaptoethanol 390 µl

1) Thaw cells.2) Innoculate 10 ml of LB amp media and grow cells overnight @ 32°C3) Aliquot 1 ml of culture for glycerol permeant, store at -80°C.4) Pour remaining preculture into 300 ml of LB broth.--> NOTE: Set aside some LB in order to zero the spectrophotometer.5) Grow cells at 32°C checking OD600 every 1/2 hour.6) When absorbance reaches 1, switch culture to 42°C to induce enzyme activity.7) Grow cells for 1.5 to 2 hours at 42°C.8) Harvest by spinning cells down at 5,000xg for 20 minutes.9) Resuspend pellet in 150 ml of TNE buffer and centrifuge at 5,000xg for 20 minutes.--> NOTE: Pellets can be frozen at this point if neccessary.10) Resuspend pellet in 150 ml of high salt buffer.11) Rupture cells using a French press two times.--> Settings for French press: 12,000 psi, 762 gauge.12) Spin homogenate at 5,000 rpm (7,000xg) for 15 minutes using the SS-34 rotor.-->This step removes any unbroken cells and large debris.13) Pellet membranes by spinning supernatant at 200,000xg for 90 minutes (48,000 rpm in Beckman Ti50.2).14) Resuspend pellet in 5 ml of TNE buffer using a Pasteur pipet, vortex and pass through 18G spinal needle.15) Bring volume up to 150 ml with TNE buffer.16) Spin at 200,000xg for 60 minutes (as above).17) Resuspend in 5 ml of TEM, homogenize 12-15 strokes in order to resuspend completely.18) Do a protein determiniation, should have a concentration of ~10 mg/ml protein.19) Aliquot membrane suspensions and store in -80°C freezer.