BIOL 1406

PreLab 8b.3

How do I dilute the eight milk fractions that were collected during the purification of α-lactalbumin?

Previous experience has shown that the protein concentrations of some of the milk fractions you collected during the purification of α-lactalbumin (Lab 8a) are likely to exceed the linear range of the Bradford assay. Therefore, those milk fractions must be diluted before they are assayed.
 

Your Turn
Calculate how many μL of each milk fraction and how many μL of dH2O are needed to make 0.1 mL of each dilution shown in the table below. Since you will need this information during lab, enter the required amounts in this table and in your lab notebook. Note that a 1% dilution is one part of the milk fraction and 99 parts of dH2O.
Example: Prepare 0.1 mL of 4% Nonfat milk
  1. 0.1 mL = 100 μL
  2. 4% = 0.04
  3. 100 μL x 0.04 = 4 μL Nonfat milk needed
  4. 100 μL  - 4 μL =  96 μL dH20 needed
 

Preparation of 0.1 mL of each diluted milk fraction
Cuvette
# 		Milk fraction (dilution) Amount of milk fraction needed (μL) Amount of dH2O needed (μL)
10 Nonfat milk (2.0 %) Check your answer. Check your answer.
11 Nonfat milk (3.0 %) Check your answer. Check your answer.
12 Pellet (3.0 %) Check your answer. Check your answer.
13 Pellet (5.0 %) Check your answer. Check your answer.
14 Whey (50.0 %) Check your answer. Check your answer.
15 Whey (100.0 %) Check your answer. Check your answer.
A Column fraction # ____ (100 %) Check your answer. Check your answer.
B Column fraction # ____ (100 %) Check your answer. Check your answer.
C Column fraction # ____ (100 %) Check your answer. Check your answer.
D Column fraction # ____ (100 %) Check your answer. Check your answer.
E Column fraction # ____ (100 %) Check your answer. Check your answer.

 


After you prepare these 11 milk dilutions, you will add 3.0 mL of Bradford’s Reagent to each cuvette and then measure the A595 values. You will then substitute these A595 values into the equation for your standard curve (prepared from the BSA dilutions) in order to calculate the protein concentrations of the diluted milk fractions. Finally, you will use the protein concentrations of the diluted milk fractions to calculate the protein concentrations of the undiluted milk fractions.
 


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