Question

You will be given 60 ul of LDH stock solution. You need to determine the protein concentration of this stock solution and then perform a serial dilution to end up with two working solution concentrations (250ug/ml and 25ug/ml). Throughout the experiment, keep your LDH on ice. A. Determining the concentration of the stock solution: We learned the proper way to handle and clean cuvettes in a previous lab. If you have forgotten, please ask the TA or Instructor for a refresher demonstration! 1. Make sure that there are no cuvettes in the spectrophotometer turret. Close the chamber lid. 2. Turn on your spectrophotometer (switch below paper roll next to power cord). After the machine has initialized (~2minutes) a list of programs should appear on the screen. 3. Using the arrow keys (bottom left side of keypad), scroll down to the “A280” program and then press <enter>. A list of program parameters should appear. 4. Press the <triangle> key (upper right side of keypad) that corresponds to the “run test command. A box should appear that instructs you to place your blank and sample cuvettes into the machine. 5. Prepare a blank: Make 1 ml of a 1:100 dilution (using water) of the provided Tris buffer in a centrifuge tube. Vortex the dilution and transfer it to a cuvette. Do not perform a serial dilution for this step. Place the cuvette into the sample “B” holder of the spectrophotometer. 6. Prepare your sample: Make 1 ml of a 1:100 dilution (using water) of your LDH stock in a centrifuge tube. The LDH stock is a concentrated protein solution; mix the stock before you pipet from it. Vortex your dilution and then transfer the it into a new cuvette. Do not perform a serial dilution for this step. Place the cuvette into the sample “1” holder. 7. Shut the lid and press <enter> and record the A280 value. Assume the spectrophotometer gave you a reading of: 0.046 A280nm 8. Given the conversion factor that lmg/ml of pure LDH gives an A280 of 1.5, calculate the concentration of the stock LDH.

my questions are the following:

A) what is the concentration of the stock solution of LDH?

B) Based upon Part B of the protocol, show your calculations to make the 250ug/ml LDH solution.

C) Based upon Part B of the protocol, show your calculations to make the 25ug/ml LDH solution.

Answer 1

Answer A:

Conc. of diluted LDH = (1 x 0.046)/1.5 = 0.030 mg/ml

Conc. of LDH = 0.030 x 100 (Included conversion factor)

                       = 3 mg/ml

Answer B:

Stock conc. (M1) = 3 mg/ml = 3000 µg/ml

Final conc. (M2) = 250 µg/ml

Volume required for final solution (V2) = 600 µl

Volume of need from stock solution (V1) = ?

According to formula of M1V1 = M2V2

V1 = M2V2/M1

      = (250 x 600)/3000

     = 50 µl

To make a 250 µg/ml solution, we need to take 50 µl of stock solution (3 mg/ml) and 550 µl of Tris buffer.

Answer C:

Now, consider the 250 µg/ml solution as stock conc. and use same formula.

V1 = (25 x 500)/250

      = 50 µl

To make a 25 µg/ml solution, we need to take 50 µl of solution B (250 µg/ml) and 450 µl of Tris buffer.