Protein Extraction and Gel electrophoresis – bacteria
(adapted from Bio-Rad’s Comparative Protein Profiler)
Step 1: Protein extraction
1. Label one 1.5 ml screwcap microtube for each of 6 bacterial samples. The bacteria are as follows
(additional bacteria can be used/substituted):
a. Citrobacter freundii
b. Azotobacter vinelandii
c. Rhizobium leguminosarum
d. Bacillus subtilis
e. Escherichia coli
f. Serratia marcescens
2. Add 250 μl of Bio-Rad Laemmli sample buffer to each labeled microtube.
3. Using a sterile inoculating loop, take a swipe of bacteria (to approximately fill the open loop –
keep consistent between samples) from a petri dish, and transfer into the appropriately labeled
micro test tube, swishing the loop around the buffer to dislodge the bacteria. Close the lid.
4. Flick the microtubes 15 times to agitate the bacteria in the sample buffer.
5. Repeat steps 3 & 4 for each bacterial sample.
6. Incubate for 5 minutes at room temperature.
7. Heat the bacterial samples in screwcap microtubes for 5 minutes at 95°C.
8. Centrifuge microtubes at maximum speed for 5 minutes. Leave the tubes at room temperature.
Step 2: Gel electrophoresis
1. Prepare a precast polyacrylamide gel cassette (Bio-Rad 12% 10 well Mini Protean TGX Precast
Gel) by removing from the package and peeling off the green tape on the bottom of the gel.
2. Remove the comb from the polyacrylamide gel.
3. Place gel cassette into the electrode assembly with the short plate facing inward. Place a buffer
dam on the opposite side of the electrode assembly, with notch on buffer dam facing inward.
4. Slide gel cassette, buffer dam, and electrode assembly into the clamping frame.
5. Press down the electrode assembly while closing the two cam levers of the clamping frame.
6. Lower the inner chamber into the mini tank.
7. Completely fill the inner chamber with 1x TGS electrophoresis buffer, making sure the buffer
covers the short plate (~150 ml). Check for leaks by waiting 1-2 minutes, and noting if the buffer
level decreases in the inner chamber.
8. If there are leaks, pour out buffer (save it!), and re-assemble the components. Repeat step 7.
9. Fill mini tank approximately 200 ml of 1x TGS electrophoresis buffer, up to the “2 gel” line.
10. Load your gel, using p20 micropipettors and gel loading tips. Be careful to not puncture the gel
with the gel tips, and to get the tip in the narrow space in the gel cassette. Practice loading the
gel with air before loading with samples:
a. Standards lane: 3 µl Precision Plus Protein Kaleidoscope prestained standards
b. Lane 1: 10 µl Citrobacter freundii
c. Lane 2: 10 µl Azotobacter vinelandii
d. Lane 3: 10 µl Rhizobium leguminosarum
e. Lane 4: 10 µl Bacillus subtilis
f. Lane 5: 10 µl Escherichia coli
g. Lane 6: 10 µl Serratia marcescens
h. Lanes 7-10: 10 µl laemmli sample buffer
11. Electrophorese for 30 minutes at 200 V in 1x TGS electrophoresis buffer.
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Step 3: Staining, Destaining, selecting protein bands
1. Remove gel cassette from clamping frame.
2. Gently remove gel from gel cassette, and rinse in distilled water in gel tray. Discard water.
3. Pour 25 ml Coomassie blue stain over gels and let stain ~1 hour. Improve staining results by
placing gel trays on a slow-moving shaker.
4. Discard stain, rinse with distilled water, and destain with fresh distilled water for 2+ hours,
changing water every ~30 minutes. Improve destaining results by placing gel tray on a slowmoving shaker.
5. Select a band on your gel that you would like identified. Using a scalpel, remove the band from
the gel, being careful to only cut around that band. Place band in appropriately labeled tube
(with sample name and group name).
Step 4: Protein in-gel digestion and MALDI-TOF preparation
(please leave for instructors to complete the procedures for you if time runs out)
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Add 200µL of 100% acetonitrile (ACN) to your gel tubes. Vortex for 5 minutes to dehydrate gel.
Remove liquid. There should be a hard gel piece remaining in the tube.
Aspire all the liquid using micropipettor with a p20 tip attached to a p1000 tip.
Add 500 µl 50% ACN : 50% ammonium bicarbonate (100 mM), vortex for 15 minutes.
Aspire all the liquid using micropipettor with a p20 tip attached to a p1000 tip.
Add 200uL of 100% ACN. Vortex for 5 minutes to dehydrate gel.
Remove all liquid. There should be a hard whitish gel piece remaining in the tube.
Add 50 uL of trypsin solution (1 ng/ul in 50 mM ammonium bicarbonate) to dried gel. Let gel
pieces swell on bench for 5 minutes.
Transfer to an incubator at 37°C overnight.
Add 50 µl 80 % acetonitrile/0.1% formic acid. Vortex for 5 min.
Briefly centrifuge and remove all liquid and transfer into a new 1.5ml microcentrifuge tube.
Label the tube clearly.
Repeat steps 11 and 12 and combine liquid in the tube.
Place sample into speed-vac and evaporate for 30-50 minutes. Samples are ready for Matrix
Assisted Laser Desorption Ionization (MALDI)-Time of Flight (TOF) Mass Spectrometry (MS)
(MALDI-TOF MS) or Electrospray Ionization (ESI)-MS/MS.
For MALDI-TOF MS, dissolve sample in 5 µl MATRIX solution and spot 1 µl on MALDI plate. Let it
dry. For ESI-MS/MS, solubilize sample in 12 µl 2% acetonitrile and 0.1% formic acid.
Protein identification by PMF
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Turn on the high voltage (lightning bolt icon) 30 min before using the 4700 MALDI-TOF/TOF.
Click on the eject plate icon (plate with a down arrow). On the dialog that has appeared click OK.
Hold the ejector arm while carefully sliding the plate into the grooves.
Click on the load plate icon (plate with an up arrow). In the dialog box that has appeared,
choose the desired spot set, then click select, and in the new dialog box click load.
5. When the machine has finished aligning the plate, the camera screen will stop moving and the
laser will be in position D14.
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Click on the spot set, and then go to job tab.
On the box Mass accuracy optimization, select default calibration.
On the queue table, click the drop down menu in the spot label column 1st row and select CAL 1
Select the whole row, right click and then click on inset multiple rows
On the dialog box that has appeared click ok.