Archives: Methods

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

Reference:

Methods in Enzymology, Vol. 125: 309-328, 1986.

Octyl-POE was obtained from Bachem.

Method:

1. Harvest cells at 7K for 15 minutes. Resuspend the pellet in 20% sucrose, 10mM Tris-HCl, DNase I (50ug/ml).
2. French press at 15,000 psi three times. Centrifuge the cell lysate at 3,000 rpm for 10 minutes.
3. Set up a 2-step sucrose gradient (50% and 70%). Apply the sample on top of the gradient and centrifuge at 21,000 rpm overnight in a SW 27 rotor.
4. Collect the outer membrane band by poking a hole at the bottom of the tube and dripping the fraction into a 60 Ti tube.
5. Dilute the fraction with distilled water to less that 20% sucrose. Centrifuge at 45,000 rpm for 1 hour.
6. Resuspend the pellet in 10mM Tris HCl pH 8, 0.5% Octyl-POE. Incubate for 1 hour at 37^oC or sonicate 3 x 10 seconds.
7. Centrifuge at 150,000 g for 1 hour.
8. Retain supernatant and resuspend pellet in 10 mM Tris-HCl pH 8, 3% Octyl-POE. Incubate for 1 hour at 37^oC or sonicate 3 x 10 seconds.
9. Centrifuge at 150,000 g for 1 hour.
10. Repeat steps 8 and 9.
11. Retain supernatant and resuspend pellet in 10 mM Tris-HCl pH 8, 50 mM EDTA, 3% Octyl-POE. Incubate for 1 hour at 37^oC or sonicate 3 x 10 seconds.
12. Centrifuge at 150,000 g for 1 hour.
13. Repeat steps 11 and 12.
14. Resuspend pellet in water.
15. Check all fractions for the protein you seek by SDS-PAGE. Opr P is primarily solubilized in 3% Octyl-POE with 50 mM EDTA.

Reference:

Ames, G.F.L. and Nikaido, H. 1976. Biochemistry. 15:616-623.

Materials:

Gel solution:

Sample preparation:

Heat for 5 minutes at 100^oC and add:

Top buffer: 0.02M NaOH (0.4 g/500 ml)

Bottom buffer: 0.01M H₃PO₄ (0.57 ml/500 ml)

Methods:

1. Prepare the gel plates as usual.
2. Mix all the gel solution components except the TEMED. Degas for exactly 2 minutes at exactly 20 psi on the old vacuum pump. Over-degassing has led to premature polymerisation in the past.
3. Add the TEMED and pour quickly into plates with the comb in place. NB: Polymerisation of these gels may often take up to an hour especially near the comb. Remove the comb carefully and don’t give up too soon.
4. Prepare samples as above. The concentration of urea in the samples has been reduced slightly from 8M because it would often “precipitate” in the syringe.
   

   While preparing samples, pre-focus the gel as follows: place top and bottom buffers in chambers of gel attach leads and run with no samples as follows

   50 V Constant voltage 15 min

   200 V Constant voltage 15 min

   300 V Constant voltage 15 min

   400 V Constant voltage 30 min

5. apply samples to gel and electrophorese at 400 V at least 20 h. (minimum of 8000 V-h).
6. Gel can either be stained or used in the second dimension (below).

Staining:

Fixer:

• 17.3 g sulphosalicyclic acid
• 57.5 g trichloroacetic acid
• 500 ml dH₂O

Stain: regular Coomassie stain (MeOH, HOAc) and destain.

Second Dimension

1. cut out gel strips about 1 cm wide
2. soak gel strip for 30 min in a petri dish in regular electrophoresis buffer (Tris, glycine) with the addition of 4% SDS
3. the second dimension gel is a regular SDS-PAGE gel. It is easier to place the gel strip on the top of the gel if the notched plate has a bevelled edge.
4. apply gel strip to top of second dimension gel making sure the edges are in close contact. Seal the gel slice with agarose at the sides and across the top to hold it in place and electrophorese as usual for SDS-PAGE.

Reference:

Fields, R. 1972. Methods in Enzymology. 25:464-469.

Materials:

Solution A: 100mls of 0.1M Na₂SO₃ (fresh each week)Solution B: 1.0l of 0.1M NaH₂PO₄

Solution C: 1.0l of 0.1M Na₂B₄O₇ in 0.1M NaOH (make up in acid and ddH₂O- washed glass).

Trinitrobenzene sulfonate (TNBS) 10g in 10ml H₂O, heat to dissolve and remove black flecks of oil by centrifugation. Add HCl to 2M and cool to room temp. Wash the crystalline precipitate on a glass filter with 1M HCl. Desiccate and store at 4^oC in brown bottle. Make up to 1.1M fresh daily (100mg recrystallized TNBS in 0.2ml H₂O).

Method:

1. Make fresh daily Solution D: 1.5ml Solution A + 98.5ml Solution B
2. Standard curve: BSA at 0.1, 0.2, 0.5, 1.0, and 2.0 mg/ml in 0.25ml H₂O.
3. Samples: same concentration range as above in same volume.
4. Add 0.25ml solution C
5. Add 10µl TNBS (take note of time!)
6. Incubate exactly 5 min at 23^oC.
7. Add 1ml solution D to stop reaction.
8. Measure OD₄₂₀. Standard curve should range from about 0.09 to >1.8

Calculation:

OD₄₂₀ = 1.0 = 52nmol amino groups = 78nmol/1.5ml assay mix

“1.0mg/ml” BSA sample (0.25mg) = OD₄₂₀ of 0.945 = 73.41nmol NH₃

therefore, 0.25 mmol BSA = 73.41 nmol NH₃ groups

67,000

3.73nmol BSA = 73.41 nmol NH₃

therefore 1 molecule BSA = 19.8 molecules of lysine.

1. Run protein sample of choice (at least 200 picomoles) on SDS-PAGE.
2. After electrophoresis, soak the gel in transfer buffer for 10-20 min.
3. Prepare the PVDF membrane (0.45µm pore size from Millipore) by first soaking it in 100% Methanol for about 3 seconds and then let it rinse in water for 5 minutes to remove the methanol. The membrane must remain wet throughout the procedure. If it does become dry then it must be re-wetted with methanol before it can be used again. The filter is then equilibrated with the transfer buffer for 10-20 minutes (can be done while the gel is soaking).
4. Assemble the cassette for transfer as follows starting from the negative electrode (BLACK):
   

   sponge wetted in transfer buffer.

   filter paper (Whatman 3 MM) moistened with transfer buffer.

   protein gel (remove bubbles between gel and the Whatman paper.

   PVDF membrane (remove all bubbles).

   moistened filter paper.

   sponge soaked in transfer buffer.

5. Insert the cassette into the blotting apparatus, making sure that the membrane is facing the positive electrode and the gel is facing the negative electrode.
6. The protein is transferred over a two hour period at 0.2A or 18V in a Bio-Rad Transblot apparatus.
7. After transfer, the PVDF is rinsed in water for 5 min to remove and glycine picked up from the transfer buffer.
8. Place the filter in Commassie blue stain for a 30 min period at room temperature on a tilt table.
9. Destain the filter for 5-10 minutes and then rinse in water for 5 –10 minutes on a tilt table at room temperature.
10. Air dry the membrane on a piece of Whatman filter paper. It can be stored by wrapping it in Saran wrap and freezing it at -20^oC.
    

    TRANSFER BUFFER	STAIN	DESTAIN
    25mM Tris	5g/l Coomassie Brilliant Blue R	50% methanol
    192mM glycine	25% isopropanol	10% acetic acid
    20% methanol	10% acetic acid
    pH to 8.3

Reference:

Palva, E.T. and Randall, L.L. (1976). J. of Bacteriol.

Method:

1. Concentrate logarithmic phase cells 100-fold to give approximately 1 x 10¹⁰ cells/ml in a volume of 1 ml. After centrifugation, outer membrane permeability should not be altered (we have tested this using a microtitre nitrocefin assay) and the cells should be motile, suggesting no appreciable disturbance of the cell surface.
2. The cells are crosslinked by the addition of 0.01 – 0.1mg/ml of crosslinking reagent, reacted for 2 minutes and the reaction is terminated with excess 1M Tris-HCl, pH 8.5, as described for purified proteins.
3. Outer membranes are isolated and analysed by two-dimensional gel electrophoresis.

References:

Reithmeier, R.A.F. and Bragg, P.D. (1977). Biochim Biophys Acta. 466:245-256.
Angus and Hancock. (1983). Bacteriol. 153:1042-51.

Materials:

1. Outer membranes (or purified proteins) at a protein concentration of approx. 2 mg/ml (500 µg/ml).
2. dithio-bis-succinimidylpropionate (DSP) stock solution; make up in DMSO at 15X the desired final concentraion (Final concentration will be approx. 2 mg/ml for outer membranes and 0.5 mg/ml for purified proteins)
3. 0.2 M Triethanolamine buffer pH 8.5
4. 1 M Tris-HCl pH 8.5
5. gel sample buffer without ß-mercaptoethanol (2-ME) (ie. 4% SDS/0.5 M Tris pH 6.8/20% gylcerol).
6. heating block at 100 ^oC
7. 11% ployacrylamide gel
8. Second dimension; soaking buffer: 0.125 M Tris-HCl pH 6.8/10% 2-ME or 0.125 M Tris-HCl pH 6.8/10% dithiothreitol.
9. 11% gel stacking gel with no wells ( poured to 3/16 from top of plate). We also have a special comb which incorporates two small wells to run molecular weight standards or a reference sample concurrently.
10. 5 ml 0.8% agarose/gel

* NOTE: – the number of gels you require for the second dimension should be made at the same time as the one for the first dimension.

Method:

First dimension:

1. outer membrane and purified protein preparations are prepared in 0.015 ml volumes of 0.2 M triethanolamine buffer pH 8.5, following the specifications of Reithmeier and Bragg (1977).
2. Add 1 µl of the crosslinker, DSP, ( dissolved in dimethylsulphoxide) to make a 1/15 dilution to give the optimun final concentration. For the first experiment, a range of concentrations should be tried.
3. React at room temperature for between 15 s and 2 min ( also determined by trial). A shorter reaction time would be preferable, to minimize fortuitous crosslinking of protein.
4. Add 5 µl 1 M Tris-HCL pH 8.5 (an excess) to stop the crosslinking reaction.
5. Dilute 1:1 into sample buffer containing SDS, Tris and glycerol without reducing agent.
6. Heat samples at 100^oC for ten minutes.
7. Electrophoresis in the first dimension as for a normal protein gel. It is best to run parallel samples for second dimension electrophoresis on one side of the gel and those for direct staining to visualize the first dimension on the other side.

Second Dimension:

1. Cut strips representing each sample well to be re-electrophored from the first dimension gel with a razor blade. We have special blades which are 5 in. long for this purpose. Make a very small notch to mark the bottom end of each cut out strip before soaking them.
2. Soak the first dimension gel strips in 10% 2-mercaptoethanol (v/v) or 10mM dithioerythritol (w/v) in a plastic petri dish for 30 min at room temperature, with intermittent agitation.
3. Lay the soaked strips carefully atop the second dimension gel, being sure to pre-moisten the glass of the gel plates with electrophoresis buffer so that the strips will slide between the plates. Using gloves and a thick spacer, nudge the strips into the gap between the plates so that the strip is in contact with the second gel at all points along its length. Mark which end is the top of the strip and which is the bottom on the plates.
4. Seal the strips atop the second dimension SDS-polyacrylamide gel with 0.8% (w/v) agarose and electrophorese as normal. A small amount of the same sample (or tracking dye) may be added if a well was incorporated into the stacking gel. This will serve as a reference point to find the protein(s) you are interested in (and/or to follow the progress of the run).

Reference:

Palva, E.T. and Randall, L.L. (1976). J. of Bacteriol.

Method:

1. Concentrate logarithmic phase cells 100-fold to give approximately 1 x 1010 cells/ml in a volume of 1 ml. After centrifugation, outer membrane permeability should not be altered (we have tested this using a microtitre nitrocefin assay) and the cells should be motile, suggesting no appreciable disturbance of the cell surface.
2. The cells are crosslinked by the addition of 0.01 – 0.1 mg/ml of crosslinking reagent, reacted for 2 minutes and the reaction is terminated with excess 1M Tris-HCl, pH 8.5, as described for purified proteins.
3. Outer membranes are isolated and analysed by two-dimensional gel electrophoresis.

This technique comes from M. Selsted and has been modified slightly. The technique is used for separating small cationic peptides. Generally, a 15% gel is used although 12% gels are not uncommon.

Recipe:

Amount for 1 large gel or two small gels

Solution B: 43% (v/v) acetic acid

Notes:

• Set up gel plates as you would protein gels
• Mix all ingredients except TEMED. Make sure all urea is dissolved.
• Add TEMED and pour the gels. For mini-gels, a stacking gel is not necessary so you can put the comb directly into the top of the running gel. For large gels, a stacking gel is recommended, so overlay the running gel with iso butanol (as done with SDS PAGE gels) and pour the stacking gel later.
• Since the solubilization of the urea in the gel mix is an endothermic reaction, it is often useful to place the gel in the warm room to enhance polymerization. If this is not done, often the wells do not form properly.
• Once the gel is set, you can pre-electrophorese it. This is necessary to remove acetate ions from the gel. The running buffer is 5% acetic acid.

REMEMBER: RUN THESE GELS IN REVERSE POLARITY, IE. RUN THEM RED TO BLACK !!!!!

For large gels, pre-electrophorese at 150V (constant voltage) overnight, and for small gels, 150V for 1-2 hours is sufficient. During this time, the current will drop to a very low level. When the current no longer drops, pre-electrophoresis is complete, and the buffer can be discarded and replaced with new 5% acetic acid. Alternatively, at this point the gels can now be wrapped in Saran Wrap ( to prevent drying out) and placed in the cold room for up to 3 weeks (give or take). To cut down on the work, it is often prudent to make 4 mini-gels, pre-electrophorese them and store them. This way you have them ready when you want.

NOTE: Make sure that you are not hogging all the spacers and gel plates.
To prepare the sample solution:

Make 5 mls of 10 M urea (dissolve completely) and treat with Bio-Rad AG501-X6 mixed bed resin (ie. add some beads, mix gently for 15 min, let the beads settle and remove the urea). For every 900 µl of treated 10 M urea, add 50 µl of water and 50 µl acetic acid to end up with a 9M urea / 5% acetic acid solution. Add a pinch of methyl green to act as a tracking dye. Freeze this solution in small aliquots to prevent cyanate production.

The samples to be loaded on the gel should be in 5% acetic acid. Mix the sample 2:1 with sample solution and load into the wells. Run large gels at 250V and mini-gels at 120-150V until the methyl green has run off the bottom of the gel. Stain as usual.

NOTE: For blots use reverse polarity

Reference:

Hanock and Benz. 1986. BBA. 860:699-707.

Purpose:

Derivitization of amino groups to remove the positive charge characteristics of the protein, for example to determine the importance of amino groups to a protein’s function.

Materials:

1. Purified protein at approximately 0.25mg/ml (diluted in 50mM phosphate buffer pH 6.8, 3mM Na azide, and, for membrane proteins, 0.1% SDS).
2. Commercial stock solution of acetic anhydride or succinic anhydride
3. Microbeaker and flea bar.
4. pH microprobe to monitor pH during acetylation reaction.
5. 5N NaOH.

Method:

1. Dilute protein sample to correct concentration as described above.
2. Using microbeaker and flea bar, set up reaction mix so that it is stirring with the pH probe inserted and pH is monitored. pH must be maintained constantly at between 6.0 and 7.0, or irreversible denaturation of the protein may result. Addition of the acetic anhydride causes a transient drop of pH, which must be corrected with 20µl volumes of 5N NaOH.
3. Add 2µl acetic anhydride from stock solution every 10 min for 60 min, then let sit for another 60 min at room temp. During this time, maintain pH as described above.
4. Dialyse vs. 1 litre of 35 mM Na-phosphate buffer pH 6.8, 3mM Na-azide, 0.1% SDS for 4 hrs, then dialyse again in the same overnight.
5. Residual amino groups can be assayed with trinitro benzene sulphonate (TNBS).