Archives: Methods

Reagents:

• CHCl₃:MeOH (2:1)
• 0.9% NaCl

Method:

1. Obtain 2 litres of cells in late log phase.
2. Centrifuge down and remove pellet to a glass container (corex tube) using a spatula.
3. Add 20 fold the pellet volume of CHCl₃:MeOH (2:1) and agitate vigorously.
4. Centrifuge at 9000 x g for 20 minutes @ 4^oC. Decant CHCl₃ phase. It should be monophasic, but if it isn’t, remove the bottom layer. (Avoid sucking up any precipitate or material floating on the lower phase.)
5. Add one fifth of the bottom layer volume of 0.9%NaCl. Mix vigorously. Recentrifuge as above. Discard upper phase and precipitate on interface.
6. Repeat step 5.
7. Reduce volume of CHCl₃ to 1-2ml by evaporating under nitrogen.
   Phosphlipids can be quantitated by phosphate assay and identified by thin layer chromatography. Store at -20ºC or preferably -70ºC.

References:

Nikaido and Roseenberg (1983) J.Bact 153: 241-252;
Yoshimura et al. (1983) J.Bact 258: 2308-2314.

Method:

1. 6.2 µmoles of lipid (ideally egg phophatidylcholine or dipalmitoyl phosphorylcholine) and 0.2 µmole dicetylphosphate are dried at the bottom of a vacuum tube under a stream of nitrogen.
2. Suspend lipid film in 0.2 ml aqueous solution of purified porin or porin containing cell envelopes. It is best to use a concentrated protein solution (< 1 mg/ml) so that most of the detergent in the protein solution will be diluted out.
   

   E. coli porins: use 5-20 µg/200 µl

   P. aeruginosa porins: use 2-10 µg/200 µl

3. Sonicate in water bath sonicator on high 15-30 secs. Suspension should turn from opaque to translucent.
4. Dry suspension in the same vacuum tubes by warming them in a 45ºC water bath while evacuating using tube connected to the vacuum pump via a CuSO4 drying tube. The drying process takes 2-3 minutes.
5. Store liposomes overnight in evacuated dessicator containing CuSO4.
6. Gently resuspend liposomes in 0.4 ml of following and leave undisturbed at RT (23ºC) for 2h then gently resuspend by shaking with hand.
   

   12 mM stachyose (E. coli) or 17% w/v Dextran T-20 (P. aeruginosa)

   4 mM sodium NAFD ( add NAOH to NAD sol. to pH 6.0)

   1 mM imidazole-NAD (pH imidazole to 6.0 with dry NAD)

7. Filter suspension through 8 um Millipore membrane filter to remove large aggregates.
8. Make up 5 mls of sugar solutions:
   

   E coli: 18 mM sugar or P aeruginosa: 40 mM sugar

   1 mM sodium NAD

   1 mM imidazole-NAD pH 6.0

   Sugars should range in MW from 150-700.

9. Measure liposome swelling by following the change in OD400 using the Perkin-Elmer spectrophotometer and the attached chart recorder. The rate is estimated from the initial slope. To do this, as quickly as possible, add 10 to 20 µl of the liposome suspension to a cuvette containing 0.6 ml of the sugar solution and turning on the chart recorder.
10. An important control are liposomes made without protein but with the same amount of detergent as added for the protein-containing liposomes.

Objective:

Method for incorporating proteins into liposomes for liposome swelling assay or as an alternative antigen presentation method.

Reagents:

• Dioleoyl Phosphatidyl Choline
• Buffer of choice / distilled water

Methods:

1. Dry 0.5 mmole of dioleoyl phosphatidyl choline under nitrogen in a disposable glass tube.
2. Evacuate in dessicator under vacuum for 30 minutes.
3. Add buffer / dH20 to required volume and scrape the sides of the glass tube to dislodge the lipid.
4. Add protein at 1 mg/ul of lipid used.
5. Vortex for 30 seconds. Sonicate twice in a bath sonicator at 7 degree for 15 sec.

This makes multilamellar vesicles that become small unilamellar vesicles (SUV) with prolonged sonication time. To make large unilamellar vesicles, use the extruder.

Reference:

Hitchcock and Brown. 1983. Journal of Bacteriology, 154:269-277

Objective:

This is a procedure analyzing LPS produced by bacteria, by using a protein-free LPS preparation and after gel electrophoresis, staining the gel using a LPS specific stain.

Materials:

• Lysing buffer:
• 2% SDS
• 4% 2-mercaptoethanol
• 10% glycerol
• 0.1 M Tris-HCl, pH 6.8

Method:

1. Grow the bacteria overnight on appropriate agar plates.
2. Harvest cells with A sterile loop and suspend in 0.9% saline to an OD₆₀₀ of 0.4 (equivalent to 200 Klett units using a blue filter).
3. Remove 1.5ml of the suspension and centrifuge in a microfuge for 1 1/2 minutes to pellet the cells.
4. Solubilize the pellets in 100ul of lysing buffer.
5. Heat the lysates to 100^oC for 10 minutes.
6. For protein digestion of the samples, add 25ug of proteinase K, solubilize in 10ul of the lysing buffer to each boiled lysate. Incubate at 60^oC for 1 hour.
7. Run the gel as usual and stain with silver (see the silver staining protocol).

(Sandermann and Stromiger. 1972. J. Biol. Chem. 247:5123-5131.)

The advantage of this assay over the Lowry assay from which it is derived, is the presence of SDS in reagent A. This allows one to perform protein assays in nearly any detergent (eg., when detergents are used in the purification of outer membrane proteins). In addition, SDS separates membrane proteins from contaminating membrane constituents and denatures the proteins allowing more reproducible results.

Solutions:

Assay:

1. Wash all glassware with distilled water to remove detergents.
2. In 13mm borosilicate glass test tubes, add 5 and 10 µl of a ten-fold dilution of outer membrane preparation.
3. Prepare a standard curve by adding 0, 5, 10, 15, 20 and 25 µl of 0.1% BSA in water to test tubes as in step 2. Make sure you prepare 2 samples of 0 BSA so you have enough sample for the reference cuvette (1 mg/ml).
4. Add 1 ml of solution C, allow to stand for 15 minutes at room temperature.
5. Add 0.1 ml of solution D and vortex the test tube immediately, allow to stand for 30 minutes at room temperature.
6. Read at 650 nm against a reagent blank.

Reference:

Hitchcock and Brown. 1983. Journal of Bacteriology, 154:269-277.

Objective:

This is a procedure analyzing LPS produced by bacteria, by using a protein-free LPS preparation and after gel electrophoresis, staining the gel using a LPS specific stain.

Materials:

Lysing buffer:

• 2% SDS
• 4% 2-mercaptoethanol
• 10% glycerol
• 0.1 M Tris-HCl, pH 6.8

Method:

1. Grow the bacteria overnight on appropriate agar plates.
2. Harvest cells with A sterile loop and suspend in 0.9% saline to an OD₆₀₀ of 0.4 (equivalent to 200 Klett units using a blue filter).
3. Remove 1.5ml of the suspension and centrifuge in a microfuge for 1 1/2 minutes to pellet the cells.
4. Solubilize the pellets in 100 µl of lysing buffer.
5. Heat the lysates to 100ºC for 10 minutes.
6. For protein digestion of the samples, add 25 µg of proteinase K, solubilize in 10 µl of the lysing buffer to each boiled lysate. Incubate at 60ºC for 1 hour.
7. Run the gel as usual and stain with silver (see the silver staining protocol).

MATERIALS:

• Proteose Peptone #2 (PP2)
• 1M Tris-HCl, pH 8.0
• 0.5M EDTA, pH 8.0
• Triton X-100
• Deoxyribonuclease I
• Sucrose (20%, 52%, 70%)

METHOD:

1. Grow H103 in 100ml PP2 in a 2 litre flask overnight at 37ºC. Innoculate into 2 litres of PP2 in 6 a litre flask the next day. Grow until an OD₆₀₀ = 0.6 – 0.8.
2. Harvest cells at 7K for 15 minutes. Resuspend the pellet in 20% sucrose, 10 mM Tris-HCl, pH 8.0.
3. Add 10ml of DNase I (50 µg/ml). French press at 15,000 psi three times. Centrifuge the cell lysate at 3,000 rpm for 10 minutes.
4. Set up a 2-step sucrose gradient (52% and 72%). Apply the sample on top of the gradient. Centrifuge at 21,000 rpm overnight in an SW 27 rotor.
5. Collect the outer membrane band by poking a hole at the bottom of the tube and dripping the fraction into a 60Ti tube. (The band closest to the bottom of the tube)
6. Dilute the fraction with distilled water to fill the 60Ti tube. Centrifuge at 45,000 rpm for 1 hour.
7. Resuspend the pellet in distilled water. Perform a protein assay.
8. Perform the solubilization as follows:2 X in 2% TX 100, 20mM Tris-HCL, pH 8.0

   3 X in 2% TX 100, 20mM Tris-HCl, pH 8.0, 10mM EDTA

9. Sonicate at a probe intensity of 35 for two 30 second bursts and centrifuge at 45,000 rpm for 1 hour for each of the solubilizations. Save all the fractions.
10. Resuspend the pellet in distilled water. Perform a KDO assay and a protein assay. Perform gel electrophoresis on all fractions.
11. Add 1mg/ml lysozyme to the OprF containing fractions and let stand for 10 minutes. Solubilize with 2% TX 100, 20mM Tris-HCl, pH 8.0.
12. Save the supernatant and pellet. Pass the supernatant over a DEAE-Sephacel column equilibrated with 0.2% TX 100, 20mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0 (column buffer).
13. Perform the column chromatography as follows:Prepare a slurry of DEAE-Sephacel by washing several times with distilled water, then column buffer. Deaerate and pour into a 30 ml column. After column is packed, equilibrate it with at least five bed volumes of column buffer.

    Apply the sample by the dry bed method.

    Elute the porin F in three stages, two bed volumes of column buffer, two bed volumes of column buffer with 0.1M NaCl and two bed volumes of column buffer with 0.3M NaCl.

    Collect 1.0 ml fractions.

14. Perform a protein assay on each fraction and gel electrophoresis on major peaks to determine where OprF is.

1. Grow 2 litres of cells OD₆₀₀ = 0.8-1.0, pellet by centrifugation in the GS3 rotor for 10 minutes at 7000rpm and resuspend in 20ml cold 20% sucrose in 10 mM Tris pH 8.0, 50 ug/ml DNase I. Incubate at least 15 minutes at room temperature before french pressing.NOTE: The cells may be frozen in 20% sucrose to enhance breakage. The 20% sucrose plasmolyses cells, resulting in better separation of the outer and inner membranes.
2. Break twice at 15,000psi in the large French Pressure cell and put broken cells on ice immediately to stop protease action.
3. Remove the cell debris by centrifugation at 3,000rpm for 10 minutes at 4^oC.
4. A one-step sucrose gradient is used. The sucrose concentrations in the steps off the gradient may also be varied depending on the degree of separation desired. Use Beckman polyallomer tubes for the gradients.
5. For crude separation of outer and inner membranes, use a 2-step gradient:
   

   For the SW 41 rotor	For the Sw 27/8 rotor
   4ml 70% sucrose	14ml 70% sucrose
   4ml 60% sucrose	14ml 60% sucrose
   4ml sample (in 20% sucrose)	9ml sample (in 20% sucrose)
   Adjust the volume of the 70% cushion to fill the tube	To increased yield of outer membranes use 50% sucrose in instead of 60% sucrose

6. Centrifuge for 4 hours or overnight at 39,000 rpm in the SW 41 rotor at 4^oC. Centrifuge overnight at 23,000 rpm in the SW 27/8 rotor at 4^oC.
7. There should be two bands for the 2-step gradient. The upper band should be reddish and the lower one should be white. The lower band is the outer membrane. Outer membranes will band at the junction between the 60% and 70% steps. Inner membranes will band at the junction between the 20% and 60% steps. Collect the membranes by poking a hole in the bottom of the tube with a hot needle, and let the sucrose drip out, collecting only the membrane band.
8. Collect the bands separately using the bottom-drip method.
9. Place each band in a 60Ti tube and add at least two volumes of distilled water to dilute the sucrose below 20%.
10. Centrifuge at 47,000rpm for 1 hour.
11. Using a syringe, resuspend the pellets in approximately 1ml.

Zwittergent 3-14 was obtained from Calbiochem. The CMC of this detergent is 0.012% (w/v), molecular weight = 363.6. All solubilization steps used 0.1% Zwit 3-14 unless otherwise indicated. Total protein concentration in any solubilization step should not exceed 10 mg/ml for maximum efficiency of solubilization. Protocol #1 will not produce as clean a preparation of proteins as will protocol #2 which involves extensive solubilizations of the outer membrane proteins.

Protocol 1

1. Prepare envelopes from whole cells by French pressing. Separate whole cells from broken ones by low speed centrifugation (5,000 x g for 15 minutes). Pellet envelopes by high speed centrifugation (150,00 x g for 1 hour).
2. Resuspend envelope pellet in an adequate volume of 0.5 mg/ml lysozyme in 20 mM MOPS pH=7.5 by sonication 3 x 10 second bursts.
3. Add Zwit 3-14 to a final concentration of 1% (w/v) and homogenize by sonication.
4. Centrifuge 150,000 x g for 1 hour.
5. Go to step 10 of protocol #2.

Protocol 2

1. Prepare envelopes as above or prepare outer membranes as per usual protocol.
2. Resuspend envelope pellet in an adequate volume of 0.1% Zwit 3-14 in 20 mM MOPS buffer, pH=7.5 by sonicating 3 x 10 second bursts.
3. Centrifuge 150,000 x g for 1 hour. (41k in 70.1 Ti or 40k in 60 Ti)
4. Retain supernatant and resuspend pellet in an adequate amount of 0.1% Zwit 3-14, 50 mM EDTA in 20 mM MOPS pH=7.5 by sonication.
5. Centrifuge 150,000 x g for 1 hour.
6. Retain supernatant and resuspend pellet in an adequate amount of 0.1% Zwit 3-14, 0.4 M NaCl in 20 mM MOPS pH=7.5 by sonication.
7. Centrifuge 150,000 x g for 1 hour.
8. Retain supernatant and resuspend pellet in an adequate amount of 1.0% Zwit 3-14 in 20 mM MOPS pH=7.5 by sonication.
9. Check all fractions for the protein you seek by SDS-PAGE. OprP is primarily solubilized in Zwit 3-14 with EDTA and OprF is solubilized in Zwit 3-14 with NaCl.
10. Load fractions containing protein of interest on Q-Sepharose column. This column is an anion exchange resin similar to the Mono-Q FPLC (Pharmacia) resin and can be used to directly scale up from the Mono-Q column. A bed volume of approximately 130 ml was used for purification of large amounts of protein (ie. preps from 100 l of cells), however, this size of column requires a large gradient volume of 5.2 l. On the large column sample loadings of between 15 and 25 mls were used (sample concentration approximately 10 mg/ml). A gradient elution with buffers: Buffer A = 0.1% Zwit 3-14, 20 mM MOPS pH=7.5, 10% methanol; Buffer B = same as A with 1.0 M NaCl. Run at a flow rate of 6 ml/min. The gradient profile was linear from 0 to 45% buffer B for the first 30% of the run followed by a plateau of 45% B for an additional 30% of the run, then increasing linearly (in approximately 10% of total run) to 100% B to elute off any remaining sample. The porin proteins tend to elute in the last major peak before increasing to 100% B.
11. Fractions containing the protein of interest are pooled and washed with buffer A in Centricon units (Millipore) to wash away the salt and concentrate the sample.
12. Sample is then applied to Mono-Q column (Pharmacia) and eluted with a linear gradient using the same buffers as above.
13. Fractions of interest are again washed and concentrated with the Centricon units and either lyophilized or stored at -70 C to prevent degradation of the proteins.

Solubilization Of Outer Membrane D₂ Protein

As per Protocol #2 but the pellets are solubilized in this series of solutions:

Step 2: 1. 1% Zwit. 3-14 in 20mM MOPS pH 7.5

Step 4: 2. 1% Zwit. 3-14 in 20mM MOPS pH 7.5

Step 6: 3. 1% Zwitt 3-14: in 20mM MOPS pH 7.5, 10mM EDTA, 0.1 m NaCl:

Step 8: 4. dH₂O

Lipid A is prepared using a modification of the method of Rietschel et al (1977).

Equimolar amounts (standardized for KDO content) of lyophilized LPS is resuspended in 15ml in 50mM sodium acetate buffer, pH 3.0. The buffer is at pH 3.0 in an attempt to lessen the chance of removing labile phosphate groups from Lipid A.The resuspended LPS is heated at 100^oC for 90 minutes, then frozen and thawed four times to promote aggregation of Lipid A.

Samples are centrifuged in thick-walled glass tubes at 9500 rpm for 5 minutes, washed once with sodium acetate buffer and washed twice more with deionized water to remove sodium acetate prior to lyophilization.

Samples are resuspended in distilled H₂O and assayed for the loss of KDO content to ensure the complete removal of LPS carbohydrate from Lipid A.