This work was supported by a studentship from The University of British Columbia (to B.V.) and grants from Cystic Fibrosis Canada and Canadian Institutes 
of Health Research (to D.P.S.). We thank Jacqueline Chung for the initial preparation of protocols.

1. Culture Growth (Days 1+2)
<ol>
	<li>Grow a starter culture: 3 ml of Luria Bertani (LB) broth in a snap top 15 ml tube, at 37 &#176;C rotator overnight. Dilute overnight growth to an OD600 of 0.6. Add 1 ml of this dilution to 100 ml of LB broth in a 250 ml Erlenmeyer flask. Incubate at 37 &#176;C with shaking at 250 rpm for 16 hr into stationary phase (SP), to better approximate, in batch culture, infection conditions and to assure that bacterial virulence factors under the control of stationary phase signal factors such as rpoS and quorum sensing, are expressed. Alternatively, bacteria grown at early SP or mid- or late-logarithmic phase can be used to gain information on the bacterial adaptation phase.</li>
	<li>After 16 hr measure the OD600 and ensure the culture has reached SP by comparison to a previously constructed growth curve under identical conditions.</li>
</ol>
2. Protein Extraction (Day 3)
<ol>
	<li>Chill all media and solutions beforehand in fridge overnight. All steps should be carried out with tubes in an ice bucket. Prechill a microcentrifuge and a Beckman Coulter High Performance centrifuge (or equivalent) to 4 &#176;C and the rotor inside (rotor=JA-20, centrifuge=J2-HS).</li>
	<li>Prepare fresh 0.1 M phenylmethanesulfonyl fluoride (PMSF, 174.19 g/mol) solution in acetone by dissolving 0.0174 g of PMSF (&#39;CAUTION&#39; PMSF is toxic and corrosive, use face shields and gloves) in 1 ml of acetone (&#39;CAUTION&#39; acetone is toxic and flammable, use face shields and gloves).</li>
	<li>Make up a PBS/EDTA/PMSF solution: 0.1 ml of 0.1 M PMSF, 0.1 ml of 0.5 M EDTA, and 9.8 ml of PBS. Check the OD of the 16 hr culture is approximately where it should be in SP.</li>
	<li>Take 35 ml of the bacterial culture and transfer to an Oakridge centrifuge tube and centrifuge the culture at 4,500 &#215; g for 20 min at 4 &#176;C.</li>
	<li>Remove supernatant to a waste container and add 35 ml of cooled PBS and resuspend the pellet. Centrifuge again at 4,500 &#215; g for 20 min at 4 &#176;C.</li>
	<li>Remove supernatant and resuspend in 1 ml of cold PBS. Transfer to sterile 2 ml Eppendorf and centrifuge 1 min on high in the microcentrifuge at 14,000 &#215; g at 4 &#176;C. Remove and discard supernatant.</li>
	<li>Add 1 ml of PBS/EDTA/PMSF solution, resuspend the pellet and transfer to a 2 ml screw cap tube (with o-ring) containing ~0.5 ml glass beads (presterilized). Add the re-suspended pellet to the tube containing the glass beads and bead bash them at 4 &#176;C for 1 min in cold-room. Do this 3x, putting the sample on ice after each bash.</li>
	<li>Centrifuge at 14,000 x g for 1 min in the microcentrifuge. Remove the supernatant and put it in a sterile 5 ml polystyrene tube.</li>
	<li>To increase the yield add 1 ml of PBS/EDTA/PMSF solution to same tube contain glass beads again. Bead bash the tube at 4 &#176;C for 1 min. Do this 2x putting the sample on ice after bash. Centrifuge at 14,000 x g for one minute on the centrifuge and remove the supernatant and add to supernatant from step 2.8. Use a 1 ml syringe and a 0.20 &#956;m filter the supernatant from the polystyrene tube to a new sterile 2 ml Eppendorf tube.</li>
	<li>At this point the samples should be assayed for protein quantity, using the Pierce MicroBCA protein extraction kit and aliquots of 200 &#956;g should be frozen at -80 &#176;C until required.</li>
</ol>
3. Sample Preparation (Day 4)
<ol>
	<li>Prepare 25 ml rehydration stock solution (8 M urea, 2% CHAPS, 2% IPG Buffer, 0.002% bromophenol blue) and store in 2.5 ml aliquots at -20 &#176;C. Add 7 mg dithiothreitol (DTT, 154.2 g/mol) just prior to use to a 2.5 ml aliquot of rehydration stock solution.</li>
	<li>Process thawed samples from step 2.10 using a 2-D Clean up kit with final resuspension in 450 &#956;l of rehydration stock solution prepared in step 3.1.</li>
</ol>
4. First-dimension Isoelectric Focusing (IEF) (Day 4)
All steps in this section are using the Ettan IGPhor IEF System.
<ol>
	<li>Set up the IPG first dimension strips: clean strip holders with strip cleaning solution and then wash with Milli-Q water, leave upside down to dry. Assign each sample to a gel strip holder number.</li>
	<li>Load the sample from rehydration step in the second wider area from the (-) end. Use clean forceps to pull gel strips out of packaging. Take out protective layer from gel strips. Line strips rough side down in a slow, sliding motion to get the strips wet along the way from (-) end to (+) end.</li>
	<li>Put damp blotting paper with sterilized water on top of electrode and under the gel strips to prevent burnout. Rinse tweezers in-between rounds with purified water and ethanol. Remove all bubbles, overlay thinly with mineral oil to prevent dry outs. Line the gel strip holders on the machine.</li>
	<li>Run 12 hr at 20 &#176;C for rehydration, 1 hr at 200 V, 1 hr at 500 V, 1 hr at 1,000 V, 0.5 hr at 4,000 V, 12 hr at 8,000 V, 24 hr hold at 300 V and can be taken out at this time.</li>
	<li>After IEF is finished, it is recommended to perform the second-dimension SDS-polyacrylamide gel electrophoresis, unless the IPG strip are being frozen at -80 &#176;C in Saran Wrap coated with mineral oil for future analysis. Alternatively, in order to avoid point streaking due to DTT access it is possible to include a second equilibration step of the IPG strips prior to the second dimension with a buffer that contains iodoacetamide.</li>
</ol>
5. Second-dimension SDS-polyacrylamide Gel Electrophoresis (Day 5)
All steps in this section are using the Ettan DALTsix electrophoresis apparatus.
<ol>
	<li>To assemble the gel caster apparatus, thoroughly clean all components and ensure that gel caster is level and the grey rubber binding is lubricated with silicon gel. Put the black rubber triangular stopper on the bottom. Then, alternately place the separator and the glass plates with the shorter plate of glass facing front. Make sure the separators are on the back and the front. Fill the remaining space with the fillers (about 5 sheets) so the top is flat. Put the front panel on with the red tip on the outside. Use clamps to tighten each side and tighten the bottom screws.</li>
	<li>Preparing the gel: Add 297.3 ml of Duracryl, (&#39;CAUTION&#39; Duracryl is toxic use face shields and gloves), 147 ml of Tris Buffer 1.5 M pH 8.8, and 143.3 ml of Milli-Q water into a flask with vacuum tip containing a stir bar. Put lid on top and mix solution at moderate speed with the vacuum on for 20 min. The vacuum is used to de-gas the solution. Make a fresh 10% of ammonium persulfate solution (APS, 218.8 g/mol): 0.2 g of APS to 2 ml of Milli-Q water.</li>
	<li>Adding the gel mixture to the gel caster: When vacuum is done add 6 ml of 10% sodium dodecyl sulfate (SDS, 228.38 g/mol) to the side of the flask to avoid putting more bubbles in the solution. Add APS, stir, add 0.25 ml N,N,N&#39;,N&#39;-tetramethylethylenediamine (TEMED, 116.20 g/mol) (&#39;CAUTION&#39; TEMED is flammable use face shields and gloves) and stir.</li>
	<li>Pour gel mixture into gel caster through a funnel inserted at the back of the apparatus. Pour up to an inch below the short plate. Add 1.5 ml of water-saturated butanol to the top of the gel and wrap top with saran wrap to avoid dehydration. Wait for 1 hr to ensure full polymerization.</li>
	<li>Checking the gel: When the gel is done (check the flask), disassemble the gel caster by a sink. Check for gel integrity while rinsing the glass plates with warm water; do not allow water to enter the plate. Pour out the butanol and rinse the top of gel twice with water to get rid of butanol. Fill the top with water again and let sit if the gels are not used right away.</li>
	<li>Preparing the strips: Prepare equilibration buffer solution from the freezer (6 M Urea, 75 mM Tris-HCl pH 8.8, 29.9% SDS, 0.002% bromophenol blue). This can be preprepared and stored in 10 ml aliquots at -20 &#176;C. Dissolve 0.1 g of DTT to a 10 ml of equilibration buffer solution. One tube can be used for two IPG strips. Place the gels of the strip face down onto the buffer and let sit for 30 min. Remember which end is which.</li>
	<li>Preparing the Electrophoresis unit (2-D separation apparatus): Add 4.5 L of 1x electrophoresis buffer (25 mM Tris Base [121.1 g/mol], 192 mM Glycine [288.38 g/mol], and 0.1% w/v SDS [288.38 g/mol]) to the running tank. This buffer can be used for up to 3x. Turn the electrophoresis unit on. Check water level in water-cooling machine and fill it up with water if it is low. Turn the machine on, press mode to check the designated temperature, which should be 10 &#176;C.</li>
	<li>Make 1 L of 2x running buffer and 1 L of 1x of running buffer and store them at 4 &#176;C. Make agarose-sealing solution: weight 0.25 g of agarose and dissolve in 50 ml of 1x running buffer. Pulse for 15 sec each.</li>
	<li>Setting up the Electrophoresis unit: Use tweezers to take out each strip and remove the excess buffer on both sides with clean paper towel. DO NOT touch the strips. Pour off the water on top of the gel and line the gel top with 1x running buffer. Use clean tweezers and place the strips on top of the long plate, with the gel side facing towards you. Add 1x buffer to the strips to lubricate it. Slide strips further down by using plastic strips. Remove all bubbles in between the strip and the gel.</li>
	<li>Add agarose-sealing solution to the top of the strip to seal it. Repeat for the rest of the strips. Place the glass plates containing the gels into gel cradle and lower into the gel tank. Ensure the 1x buffer level in the outer chamber is on the designated bottom line before putting the upper chamber on.</li>
	<li>Place the frame for upper buffer chamber on top of the glass plates and ensure it is all the way down by pressing the bottom down. Use a funnel and add the 2x running buffer into the upper chamber up to the middle line. Use a funnel and add 1x running buffer to the outer chamber until it reaches the top line. Place lid on top and turn on the Electrophoresis unit and the power pack.</li>
	<li>Run overnight at 52 V, 96 mA, 5 W. Check if the current is going by looking for bubbles on the silver wire near the top. Run the gel until the leading line is 1 cm from the bottom.</li>
	<li>Proteins can also be analyzed using equipment and reagents from other companies such BIORAD or Hoefer, according to manufacturer instructions.</li>
</ol>
6. Silver Staining of the Gels for Gel Visualization (Day 5-6)
<ol>
	<li>Prepare a fix solution 1 (800 ml ethanol, 200 ml acetic acid, and 1,000 ml of Milli-Q water in fume hood into a plastic bucket). Solution is good for 6 gels. Turn all the machines off and take out the whole middle section of the Electrophoresis unit and pull it in sink. Also put the white stand in the sink. Pull out the top tray containing the 2x running buffer.</li>
	<li>Disassemble the apparatus and remove the gels from the glass plates into fix solution 1. Gels can be identified by cutting off corners when they are removed from the glass plates, the number of corners cut corresponding to the gel order in the caster.</li>
	<li>Place the container with the fix solution and the gels on a rocker for at least 1 hr to overnight at 4 &#176;C.</li>
	<li>Transfer the gels to fix Solution 2 (20 ml of 50% glutaraldehyde, 600 ml of ethanol, 5 g of potassium tetrathionate (302.46 g/mol), 136 g of sodium acetate (82.03 g/mol), and Milli-Q water to 2,000 ml) and place on a rocker for 1 hr.</li>
	<li>Wash the gels 4x for 15 min each in Milli-Q water.</li>
	<li>Stain the gels in Silver Nitrate Solution (4 g of silver nitrate (169.87 g/mol), 500 &#956;l of formaldehyde, and Milli-Q water to 2,000 ml) for 30 min or up to 48 hr.</li>
	<li>Wash gel for 1 min in Milli-Q water.</li>
	<li>Transfer to developer Solution (60 g of sodium carbonate (105.99 g/mol), 300 &#956;l of formaldehyde, 15 mg of sodium thiosulfate (158.11 g/mol), and Milli-Q water to 2,000 ml) for 5-30 min until the gel is stained.</li>
	<li>Put the gels in Stop Solution (100 g of Tris-Base (121.14 g/mol), 40 ml of acetic acid, and Milli-Q water to 2,000 ml) for 10 min.</li>
	<li>For storage, transfer the gels to Glycerol Solution (40 ml of glycerol or 400 ml if long term storage is desired, and 1,600 ml of Milli-Q water) for 10 min and then dry them using a gel dryer.</li>
	<li>Gels can be visualized after staining using imaging systems such as a scanner/densitometer, photographed using light transilluminator or Gel imager. Image Analysis software such as PDQuest 2-D analysis from BIORAD can then be used to obtain quantitative and qualitative information from proteins in a sample.</li>
</ol>
7. Gel Silver Staining for Mass Spectrometry Analysis
<ol>
	<li>Fix the gels in 50% methanol, and 5% acetic acid for 1 hr.</li>
	<li>Wash in 50% methanol for at least 10 min to overnight.</li>
	<li>Wash in distilled water 3x for 10 min.</li>
	<li>Sensitize with 0.02% sodium thiosulfate (158.11 g/mol) 2x for 15 min.</li>
	<li>Wash 3x in CHILLED distilled water 3x for 10 min.</li>
	<li>Submerge gel in CHILLED 1% silver nitrate solution (169.87 g/mol) for at least 20 min to 1 hr at 4 &#176;C</li>
	<li>Wash 2x for 1 min with distilled water. Change tray/bucket.</li>
	<li>Develop in 0.04% formaldehyde in 2% sodium carbonate anhydrous (105.99 g/mol) and discard when it turns yellow quite quick; have three batches and be ready to change every 5 min.</li>
	<li>Wash in 5% acetic acid then store in 1% acetic acid.</li>
	<li>Rinse 1.5 ml Eppendorf tubes (to hold get spot cut-outs) with USP grade ethanol and air-dry prior to use.</li>
	<li>Clean forceps, razor blades and surfaces with 100% methanol.</li>
	<li>Spray gloves and surfaces with ethanol.</li>
	<li>Excise spots in a flow hood or clean air area. Spots can be also excised using an automated spot cutter.</li>
	<li>Put them in Eppendorf tubes and ship on ice or keep in fridge.</li>
</ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+silver+stain+and+SYPRO+Ruby+Protein+Gel+Stain+with+respect+to+protein+detection+in+two-dimensional+gels+and+identification+by+peptide+mass+profiling.">A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling.</a> Electrophoresis. 21, 3673-3683 (2000).</li></ol>

The authors declare that they have no competing financial interests.

A method for proteins preparation has been described that can extract the majority of Burkholderia proteins with good reproducibility. This is demonstrated by obtaining the same protein profile from two independent preparations performed in different days using the same bacterial culture grown in LB or YEM broth as shown in Figure 1. Extraction was efficient for bacteria grown in liquid media; however we have not tested this method for bacteria grown on plates. This method was also used for furt...

The genus Burkholderia comprises more than 62 species, Gram negative organisms isolated from a wide range of niches, and it is divided in two main clusters1,2. The first cluster includes human, animal and phytotrophic organisms, and most studies have focused on the pathogenic species of this group due to their clinical importance. The most pathogenic members are B. pseudomallei and B. mallei (which causes melioidosis and glanders respectively)3,4 and opportunistic pathogens (the 17 defined species of the Burkholderia cepacia complex, BCC)5, which cause disease in cystic fibrosis (CF) and chronic granulomatous disease (CGD)1. The second cluster, with more than 30 nonpathogenic species, includes bacteria associated with plants or with the environment, and are considered potentially beneficial to the host2.
Numerous complications emerge from bacterial infection with the pathogenic members of the Burkholderia genus, such as transmission of the pathogen between patients, spread of the disease and treatment failure because of the intrinsic or acquired resistance to antibiotics making hard to eradicate in most of the cases6-9. Therefore, gaining a clearer understanding of the basis for establishment of bacterial infection is vital to the treatment of diseases caused by these organisms. In order to gain insight into the establishment of infection, extensive investigations on the bacterial components associated with pathogenesis are needed. Studies focusing on the proteomic analysis of Burkholderia organisms using proteomic approaches described proteins that have been implicated in bacterial pathogenesis as well as changes in their proteome profiles10-16.
Protein extraction methods using sonication and freeze-thawed cycles in lysis buffer containing high concentration of urea, thiourea in combination with detergent and ampholytes has been applied in Burkholderia proteomic studies10-13. Although urea is quite efficient for protein denaturation, it can establish an equilibrium in aqueous solution with ammonium isocyanate, which can react with amino acid groups, thereby forming artifacts (carbamylation reaction)17. Therefore, it is recommended to include carrier ampholytes, which act as cyanate scavengers and avoid temperatures above 37 &#176;C17. Furthermore, to prevent any chemical interference of lysis buffer with protein quantification, the same lysis buffer can be used to generate the standard curve so that the samples and the standards have the same background10. Other methodologies involve the use of alkaline buffers and detergents with heat incubation periods17,18; however these conditions might induce changes in the proteome and some detergents are not compatible with proteomics application unless subsequent detergent removal steps are included17,18.
After adequate extraction and quantification, global protein expression of each individual protein can be studied using proteomic approaches such as two-dimensional (2-D) gel electrophoresis. This technique was first described by O&#39;Farell19 and consists in the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, and then according to their molecular weight by acrylamide gel electrophoresis in the second dimension. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources19,20. This separation technique is currently available in protein-centric approaches with the great advantage of resolving protein isoforms caused by post-transcriptional modifications or proteolytic processing. Quantitative changes can be detected by comparing the intensity of the corresponding spot after staining of the gel20. However, this technique is not suited for the identification of very large proteins, membrane proteins, extremely basic and acidic or hydrophobic proteins, and is a somewhat laborious and time-consuming technique20. New peptide-centric approaches (non gel-based) that are more robust and objective become available and can be used for quantitative comparison by differential stable isotope labeling methods such as cysteine labeling by isotope-coded affinity tagging (ICAT)21, and amino group labeling by isotope tagging for relative and absolute quantitation (iTRAQ)22. The use of a single proteomic technique might give insufficient information; therefore the use of two complementary proteomic approaches is necessary for most fully assessing changes in proteome. Nevertheless, 2-D gel electrophoresis is widely used and can be routinely applied for quantitative expression of several proteins in different organisms.
Here we describe a whole-cell protein extraction and 2-D gel electrophoresis procedures for Burkholderia species that were adapted and optimized from the GE Healthcare 2-D Electrophoresis Principles and methods Handbook 80-6429-60AC 2004 (<a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a>). The protein extraction was carried out using a bead beater with glass beads in the presence of PBS containing 5 mM EDTA (ethylenediamine tetraacetic acid) and 1 mM PMSF (phenylmethylsulfonyl fluoride). This procedure allows quantification of proteins with minimal degradation and is amendable for proteomic approaches as previously reported15,23,24. 2-D gel electrophoresis was carried out using 24 cm long immobilized pH 4-7 gradient and proteins were separated according to their isoelectric point. Then proteins were separated according to their molecular weight by SDS-polyacrylamide gel. Additionally, we described a silver staining method for visualization of spot proteins, and a silver stain method that is compatible for mass spectrometry analysis. Together these procedures can allow the identification of important proteins from Burkholderia species that could be involved in pathogenesis.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Phenylmethanesulfonyl fluoride (PMSF)</td>
 <td>Sigma</td>
 <td>P7626</td>
 <td>Toxic, corrosive</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher</td>
 <td>A18-1</td>
 <td>Flammable</td>
 </tr>
 <tr>
 <td>PBS Buffer</td>
 <td>Bioscience</td>
 <td>R028</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Fisher</td>
 <td>BP118-500 </td>
 <td>toxic</td>
 </tr>
 <tr>
 <td>Glass beads (0.1 mm)</td>
 <td>BioSpec Products</td>
 <td>11079101</td>
 <td></td>
 </tr>
 <tr>
 <td>Nalgene Oak Ridge Centrifuge tubes, polycarbonate (50 ml)</td>
 <td>VWR</td>
 <td>21009-342</td>
 <td></td>
 </tr>
 <tr>
 <td>MicroBCA protein extraction kit</td>
 <td>Pierce</td>
 <td>23235</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge Tubes 2.0 ml conical Screw cap Tubes with Cap and O-Ring</td>
 <td>Fisher</td>
 <td>02-681-375</td>
 <td></td>
 </tr>
 <tr>
 <td>2-D Clean-Up kit</td>
 <td>GE Healthcare</td>
 <td>80-6484-51</td>
 <td></td>
 </tr>
 <tr>
 <td>Urea</td>
 <td>Invitrogen</td>
 <td>15505-050</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>Amersham Biosciences</td>
 <td>17-1314-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>MPBiomedical, LCC</td>
 <td>856126</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>Immobiline DryStrips pH 4-7 24 cm</td>
 <td>Amersham Biosciences</td>
 <td>176002-46</td>
 <td></td>
 </tr>
 <tr>
 <td>Duracryl</td>
 <td>Proteomic Solutions</td>
 <td>80-0148</td>
 <td>Very toxic, carcinogen</td>
 </tr>
 <tr>
 <td>Ammonium persulfate</td>
 <td>Fisher</td>
 <td>BP179-100</td>
 <td>Flammable, toxic, corrosive</td>
 </tr>
 <tr>
 <td>N,N,N',N'-tetramethylethylenediamine (TEMED)</td>
 <td>Invitrogen</td>
 <td>15524-010</td>
 <td>Flammable</td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulfate (SDS)</td>
 <td>Fisher</td>
 <td>BP166-500</td>
 <td>Acute toxicity, flammable</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Invitrogen</td>
 <td>15510-027</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Fisher</td>
 <td>HC1100-1GL</td>
 <td>Flammable, toxic</td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Fisher</td>
 <td>A491-212</td>
 <td>Flammable, corrosive</td>
 </tr>
 <tr>
 <td>Glutaraldehyde</td>
 <td>Fisher</td>
 <td>G151-1</td>
 <td>Very toxic, corrosive, dangerous for the environment</td>
 </tr>
 <tr>
 <td>Potassium tetrathionate</td>
 <td>Sigma</td>
 <td>P2926</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>Sodium acetate</td>
 <td>EM Science</td>
 <td>7510</td>
 <td></td>
 </tr>
 <tr>
 <td>Silver nitrate</td>
 <td>Sigma</td>
 <td>209139</td>
 <td>Corrosive, dangerous for the environment</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Sigma</td>
 <td>252549</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>Sodium thiosulfate</td>
 <td>Sigma</td>
 <td>S7026</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>EMD</td>
 <td>9230</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>MPBiomedical, LCC</td>
 <td>808831</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>MPBiomedical, LCC</td>
 <td>800688</td>
 <td></td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Fisher</td>
 <td>A412-4</td>
 <td>Flammable, toxic, health hazard</td>
 </tr>
 <tr>
 <td>Sodium carbonate anhydrous</td>
 <td>EMD</td>
 <td>SX0400-3</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>ACROS</td>
 <td>415080010</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>JA-20 ultracentrifuge rotor</td>
 <td>Beckman Coulter</td>
 <td>334831</td>
 <td></td>
 </tr>
 <tr>
 <td>Mini Beadbeater</td>
 <td>Biospec Products</td>
 <td>3110BX</td>
 <td></td>
 </tr>
 <tr>
 <td>Ettan IPGphor II Isoelectric Focusing System and accessories</td>
 <td>GE Healthcare</td>
 <td>80-6505-03</td>
 <td><a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a></td>
 </tr>
 <tr>
 <td>Ettan DALT Large Vertical electrophoresis system</td>
 <td>GE Healthcare</td>
 <td>80-6485-27</td>
 <td><a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a></td>
 </tr>
 </tbody>
</table>

total protein extraction and 2-d gel electrophoresis methods for burkholderia species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Comparative analysis of the protein profiles extracted from the same bacterial culture on two different occasions showed similar patterns banding indicating successful protein extractions. Molecular weight proteins extracted ranged from 10-150 kDa. Figure 1 shows representative Coomassie blue staining gel of the whole-cell protein extractions from Burkholderia multivorans (a member of the BCC) clinical isolates grown in LB or Yeast/Manitol (YEM) broth and harvested from stationary phase.
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Watch this Scientific Journal Video about Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species at JoVE.com

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

total-protein-extraction-2-d-gel-electrophoresis-methods-for

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JoVE Journal

Immunology and Infection

28.9K Views.  University of British Columbia. Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Video: Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
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Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like receptor myeloid differentiation primary response gene 88 (TLR-MyD88) signaling pathway. IRF5 activation involves phosphorylation, dimerization, and subsequent translocation from the cytoplasm into the nucleus, which in turn induces the gene expression of various pro-inflammatory cytokines. A detection assay for IRF5 activation is essential to studying IRF5 functions and its relevant pathways. This article describes a robust assay to detect endogenous IRF5 activation in the CAL-1 human plasmacytoid dendritic cell (pDC) line. The protocol consists of a modified nondenaturing electrophoresis assay that can distinguish IRF5 in its monomer and dimer forms, thus providing an affordable and sensitive approach to analyze IRF5 activation.

A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like ...