Header image  
Biochemistry Laboratory Manual  
  HOME | RELEVANCE | BACKGROUND | PROCEDURE | PRELAB
   
 
Quantification of Lysozyme Extracts - Procedure

Part A: Protein quanitification.

  1. Turn on the spectrometer and set it to a wavelength of 562-nm. It is important to do this first thing as the instrument should be allowed to warm up for at least 15 minutes prior to taking readings.

  2. Remove your fractions, unpurified egg white protein, and column wash from the cold room and allow them to come to room temperature.

  3. Collect enough standard albumin (BSA) to prepare the 7 standard solutions you calculated in your pre-lab. Be sure to prepare each of the standards using the same low salt buffer you used in during the lysozyme elution.

  4. Pipette 100-µL of each standard and unknown sample into an appropriately labeled test tube. Be sure that your column wash and the unpurified egg white are included as unknown samples.

  5. Making sure to mix well, add 2-mL of BCA working reagent (already prepared) to each of the test tubes.

  6. After each of the test tubes have been covered well with parafilm incubate all of them for 30-min in a water bath that has been set to 37ºC.

  7. Remove the test tubes after the 30-min incubation period is up and allow the tubes to cool down to approximately room temperature.

  8. Immediately before the tubes have reached the appropriate temperature, blank the instrument with a cuvette filled only with ddH2O.

  9. Finally, read each of the test tubes at 562-nm making sure to record the absorbances accurately. This must be done within 10-min, so be efficient yet careful.

  10. Prepare a standard curve by plotting the absorbance recorded for each BSA standard versus its concentration in mg/mL. If the correlation coefficient is < 0.96, the standard solutions must be redone.

  11. As a note, the absorbances of your unpurified egg white and column wash may fall outside the range of the standard curve. If this is the case, you will have to dilute the respective sample by some amount so that its absorbance falls within the standard curve. Be careful to make note of your dilutions as this will be vital to complete an important table after the next lab period.

Part B: Lysozyme activity.

  1. Set the spectrometer to a wavelength of 450-nm.

  2. Remove your fractions, unpurified egg white protein, and column wash from the cold room and allow them to come to room temperature. Each fraction that indicated the presence of protein from last lab’s BCA Protein Assay along with the unpurified egg white and column wash will be assayed.

  3. Prepare 300-mL of 0.10 M sodium phosphate buffer and adjust to a pH of 7.0 based upon the calculations you proposed in your pre-lab.

  4. Next you have to prepare the cell wall substrate. This is accomplished by weighing out 0.075-g of Micrococcus leisodeikticus and preparing a suspension of the substrate in 12.5-mL of your 0.10 M sodium phosphate buffer.

  5. The cell wall suspension is then homogenized with 4 to 8 strokes to disrupt the cells followed by diluting the slurry to 250-mL with the remaining buffer

  6. With the cell wall substrate prepared, it is time to blank the spectrometer using ~6-mL of the remaining sodium phosphate buffer.

  7. When the spectrometer is blanked, pipette 5.5-mL of the cell wall substrate into a large test tube

  8. When ready to start, pipette 500-µL of the fraction to be assayed into the substrate test tube and record the time. Quickly mix the resulting suspension and pour into a cuvette.

  9. Record the absorbance every 15-sec for as long as the absorbance continues to change, but record the absorbances for no longer than ~5-min. Some fractions may have their corresponding absorbances stop changing as early as 2-min.

Printable PDF version of procedure available here