BIOL 1406

PreLab 8c.3

How should I dilute the milk fractions containing 2X sample treatment buffer so that no fraction has a protein concentration greater than 1.25 µg/µL?

 

 It is important to load an appropriate amount of protein into each well of your electrophoresis gel. If too little protein is loaded, it may be difficult or impossible to see many of the protein bands after the gel is stained. On the other hand, if too much protein is loaded, the bands may become too wide causing them to overlap and form a smear. In general, the optimum amount of protein to load into each well is about 20 μg.

Also keep in mind that there is a limit to the volume of sample that can fit into each well on your gel. To avoid overfilling the wells, it is best to load about 16 μL of sample into each well. This means that, ideally, 16 μL of sample should contain 20 μg of protein. Therefore, each sample that you load onto your gel should have a protein concentration of 20 μg/16 μL, or 1.25 μg/μL.
Lab set-up for SDS-PAGE  

 

 

 

 

 

 

 

 

 

 

With this in mind, examine the calculated protein concentrations of the undiluted milk fractions containing 2X sample treatment buffer.  This information can be found in the third table from Prelab 8c.2.  You should have printed a copy of Prelab 8c.2 after you finished entering your data and the results of your calculations.  There are 3 possible cases:

  1. The protein concentration equals 1.25 μg/μL. In this case, the sample has the desired protein concentration. Simply load 16 μL of the milk fraction into the well, and the well will contain the optimum amount of protein (20 μg.)
  2. The protein concentration is less than 1.25 μg/μL. In this case, the protein concentration is lower than desired. Unfortunately, there is no easy way to increase the protein concentration of your sample. The best you can do is use the sample with no dilution and load the maximum amount of sample that the well can hold (16 μL). Keep in mind that your well will have less than the optimum amount of protein, meaning some of the protein bands on your gel may be very light or invisible after staining.
  3. The protein concentration is greater than 1.25 μg/μL. In this case, the milk fraction should be diluted with 1X sample treatment buffer to produce a sample with a protein concentration equal to 1.25 μg/μL. For this dilution, calculate how much of the milk fraction with 2X sample treatment buffer and how much 1X sample treatment buffer should be combined to make 40 μL of sample with a final protein concentration of 1.25 μg/μL. After you have prepared 40 μL of the diluted sample (containing 1.25 μg/μL of protein), you will load 16 μL of this sample into the well and discard the remaining 24 μL.

Fill in the table below to show how you will dilute the milk fractions containing 2X sample treatment buffer. For a review of how to do the calculations for parallel dilutions, see Prelab 2.4.  The initial protein concentration for each fraction can be found in the third table from Prelab 8c.2. The final volume of your diluted sample should be 40 μL and the final protein concentration should be 1.25 μg/μL. In each case, your diluting solution will be 1X sample treatment buffer. For those fractions that already have a protein concentration of 1.25 μg/μL or less, simply write “no dilution necessary”.

IMPORTANT: After you have completed the table below, use the print button on your browser to print a copy of the table and then bring it to lab.  If you do not have a printer, copy the information by hand into your lab notebook.

NOTE: The answers provided in the table below were calculated using the answers provided in the third table for Prelab 8c.2. Your results for the nonfat milk should be very similar to the answer given below while results for the pellet, whey, and column fractions will show greater variability from group to group.
 

Dilution of Milk Fractions to Make 40 μL of Diluted Sample with a Protein Concentration of 1.25 μg/μL
Milk fraction Amount of milk fraction with 2X sample treatment buffer needed (μL) Amount of 1X sample treatment buffer needed (μL)
Nonfat milk Hint Check your answer. Hint Check your answer.
Pellet Hint Check your answer. Hint Check your answer.
Whey Hint Check your answer. Hint Check your answer.
Column fraction Hint Check your answer. Hint Check your answer.
Column fraction Hint Check your answer. Hint Check your answer.
Column fraction Hint Check your answer. Hint Check your answer.
Column fraction Hint Check your answer. Hint Check your answer.
Column fraction Hint Check your answer. Hint Check your answer.

REMINDER: After you have completed the table above, use the print button on your browser to print a copy of the table and then bring it to lab.  If you do not have a printer, copy the information by hand into your lab notebook.


Close this browser window to return to Blackboard and complete the practice quiz and assessment quiz.