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Contact: Mary Jo Kirisits

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1. Take a 1-mL sample of the bacterial cells to be assayed for protein.
2. If the medium containing the cells contains protein (e.g., LB), the cells will need to be washed and resuspended in 100 mM Tris-HCl (pH 7-7.5). Centrifuge the cells at 8,000 rpm for 5 minutes.  Remove the LB.  Add 1 mL of 100 mM Tris.    Centrifuge again at 8,000 rpm for 5 minutes.  Remove the supernatant, and add 1 mL of 100 mM Tris.  Vortex.
3. Aliquot three 200-mL μL samples into a microtitre plate.  I avoid using wells along the outside of the microtitre plate.
4. If samples need to be taken over time (e.g., protein content of a batch culture over time), place the microtitre plate (from step 3) into a shallow container with water and ice. Place this container in the refrigerator.  It will be easier to collect all of the samples and process them as a unit.  This should only be done if the experiment will last for <5 hours.  If the experiment is longer than 5 hours, the cells should be processed (sonicated and assayed) immediately after the sample is taken.
5. Tune the sonicator.
6. Pre-cool the cup horn in the sonicator. Add ice, and chill for 10 minutes.
7. Place Excel tape over the microtitre wells.
8. Add fresh ice to the water in the cup horn, and place the microtitre plate in the horn. Make sure that all parts of the microtitre plate are in contact with the ice/water slurry.  This will ensure efficient sonication for all samples.
9. Use the acoustical enclosure because the sonicator will be noisy.
10. At an output of 7.5, sonicate for 1 minute, and then let the horn rest for 1 minute. The rest period allows the horn and sample to cool; cavitation is more efficient at lower temperatures.  Repeat the sonication and rest cycles a total of 12 times (i.e., 24 minutes).  Several times during the procedure, add fresh ice around the microtitre plate.
11. The above lysis procedure was determined to be sufficient for aeruginosa. If another strain is being used, lysis should be verified by examining the sonicated samples under the microscope.  If necessary, additional cycles or a higher output can be used.
12. The lysed cells are now ready for the protein assay. The protein assay utilizes Coomassie^® (VWR:  PI23236; this item includes Coomassie^® and BSA), a reagent that allows colorimetric determination of the protein content.  Coomassie^® needs to be at room temperature for accurate results.  Therefore, 1-2 hours before you want to run the assay, take the Coomassie^® out of the refrigerator.  Gently invert it a few times to mix it.  Pour a small volume into a Falcon tube.  (For low-concentration samples [1-25 mg μg/mL], you will need 150 mL μL per sample.  For high-concentration samples [100-1500 mg μg/mL], you will need 300 mL μL per sample.)  Cover the Falcon tube in foil because Coomassie^® is a light-sensitive reagent.  Let it warm to room temperature.
13. Bovine serum albumin (BSA) standards can be prepared for a standard curve using the 2000 mg μg/mL stock solution. I generally prepare the following standards in 1.5-mL eppendorfs.  The diluent is 100 mM Tris-HCl.  The standards shown are for measuring protein in the high concentration range.  For our microtitre protocol, the standards between 125 and 750 mg μg/mL will be in the linear range.

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1. When the Coomassie^® is at room temperature, it should be inverted gently several times to mix. 300 mL μL Coomassie^® can be added to the wells of a microtitre plate (or 150 mL μL for the low-concentration protocol).  Prepare the number of wells necessary for all of your samples plus the standards.  It is best to not attempt to get the last little bit of Coomassie out of the pipet tip because this leads to bubbles, and bubbles will severely distort the absorbance results.
2. Add 10 mL μL of each standard to 300 mL μL of Coomassie^®. (If you are doing a low-concentration protocol, prepare standards in the 1-25 mg μg/mL range, and add 150 mL μL standard to 150 mL μL Coomassie^®.)
3. Mix the lysed cell samples by gentle aspiration. Add 10 mL μL sample to 300 mL μL of Coomassie^®. (For low-concentration samples, add 150 mL μL of sample to 150 mL μL of Coomassie^®.)  Mix the sample and Coomassie^® by gentle aspiration, but avoid bubble introduction.
4. Measure A_600nm If the samples are left to sit, aggregates will form and the results will not be accurate.
5. Subtract the blank from all sample absorbances. Plot the linear range of the standard curve, and determine the protein concentrations of your samples.