BISC411
EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL
Determination of Lysozyme Activity Standard
Prior to the start of the lab, the instructor obtains from the prep
room a culture of micococcus luteus that has been grown
by the staff. Spectrophotometers are calibrated and blanked with
water at OD450. This will be explained at the beginning of the
laboratory.
Students measure the OD450 of the culture. The ideal OD450
value should fall between 0.4 to 0.7. If the reading is too high,
dilute the culture with phosphate buffer until it falls into the
correct range. Use this dilution for the rest of the experiment.
Lysozyme activity is measured at OD 450 by tracking the decrease in optical density as the substrate’s cell wall is broken by the enzyme if it is present. Today you will determine the ideal concentration of a stock lysozyme solution to use as a positive standard in this assay in the future. This will also give you experience doing the assays which will facilitate future work that you will do.
You will be provided with a stock solution of lysozyme at 1 mg/ml
concentration.
Zero the spectrophotometer using a water blank. Fill the cuvette
with 3 ml of the culture at the dilution you determined to be closest
to the
ideal starting OD450. Record the starting OD450. Quickly add 0.1 ml of
the lysozyme, mix by inverting the cuvette, and immediately
place
it back in the spectrophotometer. BE CAREFUL. Once you add the lysozyme
solution to the culture the OD will fall quickly. Be prepared to take
readings
every 15 seconds. Repeat with other lysozyme dilutions similarly.
The
ideal change in OD450 is .02-.04 per minute. Continue the readings for
at least 10 readings (about 2.5 minutes).
Now, determine how dilute the lysozyme can be before it is unable to be detected by this method. To determine this dilute the stock lysozyme 1/10, 1/25, 1/50, and 1/100 or until you lose any measurable activity.
We will analyze the results of each group to determine the
sensitivity
of this assay.