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°C
3) 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.
