The overall goal of this procedure is to generate a two dimensional map of a bacterial proteome. This is accomplished by first growing a bacterial culture and harvesting the proteins. Next harvested proteins are separated in a single dimension according to their pH.
By isoelectric focusing after the first dimension separation, the proteins are further separated in the second dimension according to their molecular weight. Finally, the second dimension gel is stained and the proteins are visualized. Ultimately, results can be obtained that show differences in protein expression through two dimensional electrophoresis.
This method can help answer key questions in the bacterial pathogenesis field, such as which proteins are produced under certain conditions, and how do different bacterial isolates differ in protein production? This information can help identify candidate proteins that may be involved in virulence. After growing a 100 milliliter bur cold dairy culture to stationary phase, transfer 35 milliliters to a centrifuge tube and spin at 4, 500 Gs and four degrees Celsius for 20 minutes.
Resus, suspend the pellet in 35 milliliters of cold PBS and repeat the centrifugation. After Resus suspending the pellet in one milliliter of cold PBS transfer the solution to a sterile two milliliter micro tube and spin again. Repeat the wash in one milliliter of cold PBS before spinning at four degrees Celsius and 14, 000 Gs.For one minute, discard the supernatant and add one milliliter of P-B-S-E-D-T-A-P-M-S-F solution transferred the dissolved pellet to a two milliliter screw cap tube containing about 0.5 milliliters of pre sterilized glass beads.
Then in the cold room to disrupt the cells, use a mini bead beader three times for one minute each placing the sample on ice after each round centrifuge for one minute before transferring the supernatant into a sterile five milliliter polystyrene tube. To increase the yield, repeat the bead bashing two times with an additional aliquot of P-B-S-E-D-T-A-P-M-S-F. Pull the samples.
Then after assaying for protein quantity, store 200 microgram aliquots at minus 80 degrees Celsius until ready to use to carry out first dimension. Isoelectric focusing or IEF begin by using strip cleaning solution to clean the strip holders. Follow by rinsing the strips with milli Q water.
Then leave them upside down to dry after drying, assign each sample to a gel strip holder number. Load a previously prepared 200 microgram protein sample into the lateral well of the strip holder, and use the pipette to distribute the sample along the holder using clean forceps. Pull the gel strips out of the packaging.
Then remove the protective layer from the gel strips before lining them rough side down in a slow sliding motion to get the strips wet from the negative end to the positive end. To prevent burnout, place blotting paper dampened with sterilized water on top of the electrode and under the gel strips. Remove air bubbles and overlay with a thin layer of mineral oil to prevent dry outs.
Line up the gel strip holders on the IEF machine. Run the samples using the program listed here. The gels can be removed at any time during the last step.
When the program is complete, remove the strips from the strip holders and unless being used immediately, cover in plastic wrap and store at minus 80 degrees Celsius until ready to perform second dimension SDS page After assembling the gel castor apparatus for SDS page, prepare the gel solution in a flask with a vacuum tip and a stir bar by adding draquel 1.5 molar tris buffer and MCU water mix and degas the solution at moderate speed with the vacuum for 20 minutes. When the vacuum is finished, add 10%SDS to the gel solution by pipetting it down the side of the flask. To avoid introducing bubbles, then add freshly prepared ammonium per sulfate and temid and stir.
Pour the gel and allow it to set then dissolve DTT into previously prepared equilibration solution. Remove the strips from minus 80 degrees Celsius storage, unwrap them and place the IPG strips gel side down in the buffer and incubate for 30 minutes. Next, add 4.5 liters of electrophoresis buffer to the running tank.
Then using tweezers, remove the strips from the e equilibration solution and use a paper towel to drain the excess buffer. Disassemble the gel caster apparatus and wash the glass plates with warm water. After replacing the water on the top of the gel with running buffer, use clean tweezers to place a strip on top of the long plate with the gel side facing toward you.
Lubricate the strip with one x buffer and use a plastic strip to slide the IPG strip further down against the gel, removing all air bubbles between the strip and the gel. Add agarro sealing solution to the top of the strip to seal it in place. Then after repeating the process for all of the strips, lower it into the gel tank.
After ensuring that the one x buffer level in the outer chamber is at the designated level, attach the upper chamber. Place the frame for the upper buffer chamber on top of the glass plates and press down. Add the two x running buffer into the upper chamber up to the middle line.
Then add the one x running buffer to the outer chamber until it reaches the top line. Place the lid on top and turn on the electrophoresis unit and power pack. Run the samples overnight at 52 volts, 96 milliamps, and five watts.
Run the gels until the leading lines are one centimeter from the bottom. After runtime, remove the plates from the castor. Once the gels are removed from the glass plates, fix them in solution one for at least one hour or overnight at four degrees Celsius.
Then transfer the gels to fix solution two and rock for one hour. Wash the gels in Milli Q water four times for 15 minutes each. Then use silver nitrate solution to stain the gels for 30 minutes or up to 48 hours.
After washing the gels for a minute in Milli Q water, transfer them to developer solution until the gel is stained. About five to 30 minutes to stop the reaction, transfer the gels to stop solution for 10 minutes. Shown here is a Kumasi Blues stain gel of whole cell protein extractions from ol dia multivorans.
Clinical isolates grown in either LB or yeast mannitol broth and harvested at stationary phase. Comparative analysis of the protein profiles extracted from the same bacterial cultures on two different occasions showed similar banding patterns indicating successful protein extractions. In this figure 2D gel electrophoresis analysis of 200 micrograms of total protein from Bural dio, pseudo shows an abundance of more than 500 spots that can be individually identified by mass spectrometry.
Following this procedure, other methods like cytometry can be performed in order to determine quantitative and quantitative differences between closely related bacterial isolates. Additionally, with a modification to the staining method, investigators can determine the identity of protein spots by spot excision and mass spectrometry.
