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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.

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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 pippetting). 

1) Figure out your plan for making each solution required in the protocol.  Be certain you FINISH with the required volumes of EACH solution

  1. Calculate what is needed to make the 4 mg/ml solution
  • Calculate what is needed to make the 400 ug/ml working solution
  • Calculate what is needed to make the 40 ug/ml working solution

 Reaction Rate as a Function of Substrate Concentration

Reaction mixture:

Assay                             6                          7               8                        9                      10                            11

Lactate (mM) 9 3.15 1.8 1.35 0.9 0.45
1.5 M Tris  pH 8.8 345 ml 735 ml 825 ml 855 ml 885 ml 915 ml
15 mM Lactate 600 210 120 90 60 30
38 mM NAD 30 30 30 30 30 30
250 mg/ml LDH 25 25 25 25 25 25

2) Looking at the “Reaction Rate as a Function of Substrate Concentration” section in the lab protocol, which assay would you predict to have the highest LDH activity and why?

 
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