ISOLATION OF CYTOPLASMIC AND OUTER MEMBRANE FRACTIONS
(Method by Osborn et al., 1972, J. Biol. Chem. 247: 3962-3972)
What is needed:
Reserve the shaker.
Need 1L baffled flasks with media autoclaved in them. Rinse the flasks 6 times with
distil water before use. Examine for dirt.
Need 6 each of 500ml centrifuge bottles.
Need ultra centrifugation tubes/ bottles for T45i , SW31 and T75i rotors
Rotors are kept in K155 cold room. Inspect their o rings. If damaged, replace them. If
dry, apply SILICON grease (NO other grease please).
Locate where lysozyme, DNAse and RNAse are kept.
Make all the buffers
(note that the pH of the buffer can ranges from 7-8. It does not matter at the
end).
Stocks needed: Locate or make these before starting the procedure.
1M Tris-HCl, pH 8
1M MgCl2,
25mg/mL lysozyme (fresh)
0.5M EDTA, pH 8.
Ice cold water, 1L.
20% sucrose, 1mM EDTA, pH 8, 10mM Tris, pH 8
73% sucrose, 1mM EDTA, pH 8, 10mM Tris, pH 8
53% sucrose 1mM EDTA, pH 8, 10mM Tris, pH 8
10X PIC (protease inhibitor tablets)
2mg/mL DNase
10mg/mL RNase
0.5M sucrose, 40mM Tris-HCl, pH 8
0.25M sucrose, 20mM Tris-HCl, pH 8
10mM Tris, pH 8
10mM Na-EDTA, pH 8.
Inoculating bacteria:
consider OD 1= 5X10^8 for both Pseudomonas and Acinetobacter, and 10^9 for
Salmonella or E. coli. If starting the procedure in the morning, inoculate at 0.02.
With 4 doublings, you should get into around 0.3 OD.
If starting the procedure early in the morning, inoculate the bacteria in the late
afternoon at 4pm to an OD of 0.0000001 (or use an OD depending on the growth
rate)
For polymyxin or C18G treatment, add the compound at OD= 0.2.
For CAMP experiments, harvest at 60 to 90 min post exposure.
Media, LB, minimal or BHI: To prevent autolysis, add MgCl2 to 1mM for growing
Acinetobacter.
1. Inoculate 1L with resuspended bacteria from the plate or from the ON
culture- grow at 37°C until it is grown enough for the experiment.
Note: the volumes and concentrations of the buffers are formulated assuming
the final OD of a 1L culture should be not more than OD600 is 2)
2. Centrifuge cells at 8000g, 10 min, 4°C in the Sorvall (room K113) or Beckman
(room K110) using a 500ml Nalgene bottle. Pour off media and invert bottles
onto a paper towel to remove residual culture medium.
3. Resuspend cell pellet rapidly in 25mL ice cold hypertonic solution (0.5M
sucrose, 40mM Tris-HCl, pH 8). Make sure it is well suspended! Plasmolysis
occurs at this stage. Add a small stir bar and stir.
4. Add 200μL of freshly prepared 25mg/mL lyzozome (final concentration:
200μg/mL) and incubate on ice for 5min and stir.
5. Form spheroplasts by diluting suspension with 20ml cold 5mM Na-EDTA, pH
8 and stir for 10min (spheroplasts are usually formed in <10min when the
lysozyme destroys the cell wall). Bacteria are susceptible to lysis if the
medium become hypotonic at this stage.
Note: EDTA is necessary for maximum activity of lysozyme. It enables
permeabilization of the OM so that the larger lysozyme can penetrate the envelope.
Tris also enhances the permeabilization of the envelope.
Note: FOR PSEUDOMONAS, DON’T ADD EDTA. Instead, add 25ml ice cold water.
EDTA causes excess cell lysis without making spheroplasts.
Note: For monitoring spheroplast formation, add 40ul of spheroplasts into 1ml
water in a plastic cuvette and read the OD600. Plot the data. Reduction of OD600
denotes spheroplast formation.
6. Stabilize spheroplasts by adding 5mL 1M MgCl2 (final concentration: ~20mM
Mg2+). Mix by swirling. Bacteria are now resistant to lysis. Decant the
bacteria to a 50ml conical.
7. While spheroplasts are spinning, make the following buffer: Into 1ml of
0.25M sucrose, 10mM Tris buffer, crush 2 tablets of PIC in 2ml buffer,
dissolve well, add 100uL 2mg/mL DNase and 20uL of 10mg/mL RNaseA and
25ul of 1M MgCl2 and bring the volume to 25ml. If you have X number of
samples, make a stock for all the samples.
8. Centrifuge spheroplasts at 8000g, 10min, 4°C (in a benchtop centrifuge).
Remove the supernatant out and drain well.
9. Resuspend spheroplasts in 25 mL of this buffer
10. Lyse the spheroplasts by running them 3X in Avastin homogenizer (Xena)
Then sonicate briefly using the small tip sonicator in K129. Use the “5” setting in the
dial. Sonicate 45 second burst with in between cooling 6 times.
11. Add 250ul 0.5M EDTA to the lysate to chelate Magnesium. Centrifuge the
lysates at 8,000g, 10min, 4°C in Beckman Allegra in K126 or Eppendorf in
K155 to remove intact cells. Save the supernatant into a new conical. Discard
the pellet.
Note: if the OD of the starting culture is >1.5, then the cell density maybe too high to
be centrifuged in a 50ml conical. Then dilute the cells and centrifuge. Do this 2X.
Then filter through a 5microne (use the Millipore filtration device in K161) and then
an acrodisc filter to remove cell debris.
12. Centrifuge the supernatant fraction at 35K, 1hr, 4°C in Ti 45 rotor in the
ultracentrifuge. (STOP POINT: Alternatively you may spin overnight at 35K)
or freeze at -80C.
NOTE: There are three O rings in the Ti45. One on the bottom of the lid, one on
the top lid fastner and one inside the centrifuge bottle. Failure to inspect (and
grease or replace) each of these O rings will result in tube failure and sample
loss. There are two O rings in the Ti70 rotor as well.
Never spin the ultra centrifuge more than 35K for the Ti45 rotor. If the tube
crashed inside the Ti45, you will have to autoclave it.
13. Resuspend the membrane pellet in 4mL 20% sucrose (w/w), 1mM EDTA, pH
8, 10mM Tris, pH 8, with down homogenizer in a ultra centrifuge tube that is
use for Ti70 rotor.
14. Set up sucrose gradient in ultra-clear SW41 ultra-centrifuge tubes by adding
solutions carefully and slowly in the following order (all solutions at 4°C):
(see the method of making the gradient and also how to balance tube
weights at the end of this article).
2mL 73% sucrose
4mL 53% sucrose
~2mL total membrane in 20% sucrose (or ½ total membrane)
~4mL 20% sucrose (or to top of tube)
15. Centrifuge at 37K, ≥12hrs at 4°C in the SW41 rotor in the ultra-centrifuge.
Longer centrifugation time results in better separation (best separation is
after 48 hours).
16. Carefully collect the inner membrane fractions with a Pasteur pipette.
17. Pierce the bottom of the tube with a needle, let the buffer drain by drop wise
(control the flow by using the thumb to close the top of the tube). When the
outermembrane ring is at the hole, let it drop into a 15ml falcon tube.
***Membranes can be stored at this point at 4C or -80C***
REMOVAL OF SUCROSE AND DETERMINING YIELD OF MEMBRANE
FRACTIONS
18. Add approximately 1mL membranes to 6mL 10mM Tris, pH 8, 1mM EDTA
buffer. Mix with a Teflon stick.
19. Centrifuge at 50K, 1hr, 4°C in the Ti70 rotor in the ultra-centrifuge. Decant
the supernatant.
20. Resuspend membranes in ~700μL 10mM Tris, 1mM EDTA buffer and mix
with Teflon sticks. Homogenize with Teflon plunger.
21. Store membranes on ice and use the PIERCE Coomassie Protein Assay to
determine the protein concentration of the membrane fractions (1μL, 2μL,
and 5μL samples).
22. Aliquot the membranes and quick-freeze them in a liquid nitrogen. Store the
membranes at -80°C.
***ALL SOLUTIONS SHOULD BE STERILE AND AT 4°C
Sucrose density gradient. The gradient is prepared by layering progressively less
dense sucrose solutions upon one another; therefore the first solution applied is the
73 % sucrose solution. A steady application of the solutions yields the most
reproducible gradient. Firstly the Beckman tube is held upright in a tube stand. Next
a 200 μl pipettor tip is placed on the end of a 1000 μl pipettor tip. Both snugly fitting
tips are held steady by a clamp stand and the end of the yellow tip is allowed to
make contact with the inside wall of the tube as shown below. Now sucrose
solutions can be placed inside the blue tip and gravity will feed the solutions into the
tube slowly and steadily, starting with the 73% solution. if the solutions fail to feed
down through the tips and into the tube a small amount of positive air pressure can
be applied to the blue tip to start the flow. This is done by gently tapping on the
wide end of the blue tip. Once the 73 % solution has drained into the tube, the 53 %
solution can be loaded into the blue tip which will then flow down the inside of the
tube and layer on top of the 73 % solution.
References:
(i) LYSIS OF GRAM-NEGATIVE ORGANISMS AND THE ROLE OF VERSENE*
ROY REPASKE. Department of Bacteriology, Indiana University, Bloomington, Ind.
(U.S.A,)
VOL. 30 (1958) BIOCHIIMICA ET BIOPHYSICA ACT. Page 225
(2) HOW DOES LYSOZYME PENETRATE THROUGH THE BACTERIAL
OUTEK MEMBRANE? BERNARD WiTHOLT, HARM VAN HEERIKRUIZEN and LOE DE
LEIJ. Biochimica el Biophysica Acta, 443 (1976) 34-44
(3) Osmotic Pressure in Escherichia coli as Rendered Detectable by Lysozyme
Attack. PAUL SCHEIE. J of Bacteriology, May 1973, p. 549-555; Vol. 114, No. 2
(4) Mechanism of Assembly of the Outermembrane of Salmonella typhimuriu:
Isolation and characterization of cytoplasmic and outer membrane. M. J. Osborn, J. E.
Gander, E. Parisi JBC 1972, 247,:3962
Table 2. Relative Centrifugal Fields for the Type 45 Ti Rotor.
Entries in this table are calculated from the formula
RCF = 1.12r (RPM/1000)2
and then rounded to three significant digits.
Rotor
Speed
(rpm)
Relative Centrifugal Field (x g)
k
Factor*
At rmax
(103.8 mm)
At rav
(69.8 mm)
At rmin
(35.9 mm)
45 000
41 000
40 000
235 000
195 000
186 000
158 000
131 000
125 000
81 400
67 600
64 000
133
160
168
35 000
30 000
25 000
142 000
105 000
72 700
95 800
70 400
48 900
49 300
36 200
25 100
220
299
430
20 000
15 000
10 000
46 500
26 200
11 600
31 300
17 600
7 810
16 100
9 050
4 010
672
1 195
2 689
*Calculated for all Beckman Coulter preparative rotors as a measure of the rotor’s relative pelleting efficiency
in water at 20°C.
Figure 1. Arranging Tubes in the Rotor. Two, three, four, or six tubes can be centrifuged per run if they are
arranged in the rotor as shown.
Polycarbonate Bottles: The capped polycarbonate bottles may be centrifuged completely
filled, or partially filled (not less than half full). Again, all opposing containers for a run
must be filled to the same level. Be sure to note the reductions in run speed shown in Table 1
if bottles are partially filled.
How to pour the discontinued density layers:
The heaviest concentration is loaded first, followed by successively lighter
concentrations. This method is acceptable for wettable tubes; however, loading a
nonwettable tube (such as Ultra-Clear, polypropylene,† and polycarbonate) by
allowing solutions to run down the side of the tube can cause mixing. Gradients in
nonwettable tubes can be prepared using a gradient former by placing a long
syringe needle or tubing to the tube bottom and reversing the gradient chambers. In
that way the lightest gradient concentration is loaded first, underlayed by
increasingly heavier concentrations.
Balancing the swing buckets:
How do you balance the sample tubes, with or without buckets? The correct procedure is
without, because slight differences in bucket weight are used to balance the rotor. This is
why each rotor should always be used with "its" buckets, and the buckets always be put in
the correct place on the rotor (numbers on rotor and bucket should agree). If you add
balanced tubes (to within 1 mg) to a balanced rotor/bucket system, precession will be
minimal. This will not only improve your samples, but also reduce wear and tear of the
centrifuge drive. 1 mg may sound little, but at 260,000*g the centrifugal force will be as high
as the gravitational force is on a mass of 260 gram.