BIOL 3380 Name:_____________________________________
Circle Session: T-AM T-PM W-AM W-PM R-AM R-PM F-AM F-PM
Experiment 9 – Pre-lab Homework
Enzyme Kinetics of LDH
This pre-lab homework assignment is due at the beginning of your lab session.
You are provided with the following portion of a protocol:
 Determine concentration of enzyme stock solution, if unknown, by taking an A280 nm reading of
a 1:100 dilution (in water). Use a total volume of 1 ml in the cuvette.
 Dilute some of the enzyme stock with buffer A to make a 4 mg/ml solution.
 Serially dilute the 4 mg/ml solution with buffer A to make working solutions of 400 µg/ml and
40 µg/ml.
 Prepare 30 µl of each working solution for every sample
The PI of the lab gives you a tube of enzyme and tells you the following before disappearing into the
office to write more grant proposals:
 There is 50 µl of enzyme stock solution. The enzyme is expensive to purify, so follow the
protocol exactly, using as little of the stock solution as possible.
 The concentration of the stock solution is currently not known, but a 1 mg/ml concentration of
the pure enzyme has an A280 nm of 2.0.
 You’ll be performing the assay on 12 samples.
 Make enough of each working solution so that you have at least 400 ul to work with when you
do the assay (to cover any waste and/or inefficiencies in pipetting).
Using the spectrophotometer to read the absorbance at 280 nm, you get a reading of 0.784.
1. (2 pts) What is the concentration of the solution in the cuvette? What is the concentration of the
stock solution? (Show your work, box your answers.)
A1=2.0nm Dilution 1:100
C1=1mg/ml 0.392mg/ml(100)=
A2=0.784 
A1/C1=A2/C2 | Stock Concentration=39.2mg/mlA1 X C2= A2 X C1÷
(0.784nm X 1 mg/ml) / 2.0nm
C1(cuvette concentration) = 0.392 mg/ml
2. (1 pt) Calculate the minimum amount of each working solution would you need in order to do the
number of assays requested.
9-1
Making 12 samples, each must be 30ul
12 x 30ul = 360 ul
Minimum solution requirement = 360ul
How much excess has the PI instructed you to make?
Minimum= 360ul
Instructed amount= 400ul
400ul-360ul= 40ul
PI instructed to make additional 40ul of working solution
3. (6 pts) Figure out your plan for making each solution required in the protocol. Be certain you
FINISH with the required volumes of EACH solution!
a. Calculate what is needed to make the 4 mg/ml solution
C1V1=C2V2
39.2mg/ml(x)= 400ul(4mg/ml)
0.0392ug/ul(x)=400ul(0.004ug/ul)
-To make 4ug/ul, you need 40.8ul of solutio