Lactate Dehydrogenase  Measurements

This assay will measure the activity for the following reaction:

                                         LDH
                  Pyruvate + NADH  ------------------  L-Lactate + NAD

1. 	First prepare your cuvettes.
	Add the specified amounts of each of the following solutions to each cuvette. 
	The solutions are labeled with orange tape in the refrigerator.

	250 ul	100mM Triethanolamine HCl/ 10mM EDTA Buffer
	10  ul	10mg/ml NADH
	230 ul	H2O
	 10 ul  NADH

Fill one cuvette with 500 ul buffer. This is your "blank." Insert your cuvettes 
GENTLY into the slots, with your blank in the slot furthest away from you, 
followed by three more. They must all be oriented in the same direction to get
an accurate reading.

2.  	Program the spectrophotometer as follows:

	Push : VIS to turn on the light source
	Push: PROGRAM
	Then push: STEP until a 6 appears
	Then push: R/S
	Then: 0 + ENTER
	Then: 	  ENTER
	Then: 4 + ENTER
	Then: 1 + ENTER 
	Then: 1 + ENTER
	Then: 30 + ENTER
	Then: 5 + ENTER
	Then: 1 + ENTER
	Then: 0 + ENTER
	Then: 0 + ENTER
	Then: 1 + ENTER
	Then: 1 + ENTER
	Then: 0 + ENTER
	Then: R/S


3. 	When you no longer see "Cal" on the screen you are ready to run your samples. Make 
	sure the printer is turned on and ready to print.Quickly but carefully add 10ul of LDH
 	from the eppendorf tube labeled "LDH #1"to each of your cuvettes except the blank. 
	Make sure the enzyme is not stuck on the side of the cuvette. Give each cuvette a
	quick stir with the stir rod, close the lid, and press R/S. It should begin printing right away.

4. After 5 minutes you can print your results as follows:
	Press: 1 + ENTER
	Then: ENTER
	Then: ENTER
	Then: ENTER
	Then: ENTER
	Then: 2 +ENTER
Then: ENTER
	Then: ENTER
	Then: ENTER
	Then: ENTER
	Then: 3 + ENTER
Then: ENTER
	Then: ENTER
	Then: ENTER
	Then: ENTER
	Then: 4+ ENTER
Then: ENTER
	Then: ENTER
	Then: ENTER
	Then: ENTER
	Then: 0 + ENTER

5. Now remove all of your cuvettes except the blank and repeat steps 2 through 4 using your other two samples of LDH.