BIOL 1406

PreLab 8c.2

What are the protein concentrations of your milk fractions?


During Lab 8b, you used the Bradford assay to determine the protein concentrations of your milk fractions. You will report your results in the 3 tables below.

IMPORTANT: After you have completed all 3 tables, use the print button on your browser to print the tables and then bring them to lab. You will need this information to complete the next section of the Prelab and you will also need it during lab. If you do not have a printer, copy the information by hand into your lab notebook.

In the first table, report the A595 values measured during Lab 8b for all 11 diluted milk fractions. Also calculate and report the protein concentrations for the diluted fractions whose A595 value fell within the linear range of your standard curve. If there are any diluted fractions whose A595 value fell outside the linear range of your standard curve, simply write “NA” in the column labeled “Calculated Protein Concentration (μg/μL)”.

NOTE: The answers given below are examples of typical results. Keep in mind that results will vary from group to group and your answers will not be exactly the same as the ones provided. However, if you did the Bradford assay and calculations correctly, the protein concentrations of your 2 nonfat milk samples should be very similar to the answers given below. Values for the pellet, whey, and column fractions depend on your technique during the purification process and will show greater variability from group to group.

Protein Concentration of Diluted Milk Fractions
Milk Fraction (dilution) A595 Value (measured during Lab 8b) Calculated Protein Concentration (μg/μL)
Nonfat milk (2.0 %) Hint Check your answer.
Nonfat milk (3.0 %) Hint Check your answer.
Pellet (3.0 %) Hint Check your answer.
Pellet (5.0 %) Hint Check your answer.
Whey (50.0 %) Hint Check your answer.
Whey (100.0%) Hint Check your answer.
Column fraction (100 %) Hint Check your answer.
Column fraction (100 %) Hint Check your answer.
Column fraction (100 %) Hint Check your answer.
Column fraction (100 %) Hint Check your answer.
Column fraction (100 %) Hint Check your answer.


In the second table, report the protein concentrations of your undiluted milk fractions. As explained in Lab 8b, you can calculate the protein concentration of each undiluted milk fraction by taking the protein concentration of the corresponding diluted milk fraction and dividing it by the dilution percent. For example, if a 1% dilution of nonfat milk had a protein concentration of 0.380 µg/μL, then to calculate the protein concentration of the undiluted nonfat milk simply divide 0.380 µg/μL by 1% (0.01). This means the undiluted nonfat milk had a protein concentration of 38.0 µg/μL.

Remember that when performing the Bradford assay, you used 2 different dilutions for 3 milk fractions: the nonfat milk, the pellet, and the whey. In each case, you should calculate protein concentration using only the dilution that produced an A595 value that lies within the linear region of your standard curve. If both dilutions of a given milk fraction gave an A595 value that lies within the linear region of your standard curve, then calculate the protein concentration of the undiluted fraction using both dilutions and average the 2 answers.

NOTE: The answers provided in the second table (below) were calculated using the answers provided in the first table (above). 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.
 

Protein Concentration of Undiluted Milk Fractions
Sample Type Calculated Protein Concentration (μg/μL)
Nonfat milk Hint Check your answer.
Pellet Hint Check your answer.
Whey Hint Check your answer.
Column fraction Hint Check your answer.
Column fraction Hint Check your answer.
Column fraction Hint Check your answer.
Column fraction Hint Check your answer.
Column fraction Hint Check your answer.


During Lab 8b, you prepared your undiluted milk fractions for electrophoresis by mixing 50µL of each undiluted milk fraction with 50µL of 2X sample treatment buffer. This effectively dilutes the protein concentration of each sample in half. Taking this into account, calculate the protein concentration of each undiluted milk fraction after the 2X sample treatment buffer was added, and enter the results in the table below.

NOTE: The answers provided in the third table (below) were calculated using the answers provided in the second table (above). 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.
 

Protein Concentration of Undiluted Milk Fractions After the 2X Sample Treatment Buffer was Added
Sample Type Calculated Protein Concentration (μg/μL)
Nonfat milk Hint Check your answer.
Pellet Hint Check your answer.
Whey Hint Check your answer.
Column fraction Hint Check your answer.
Column fraction Hint Check your answer.
Column fraction Hint Check your answer.
Column fraction Hint Check your answer.
Column fraction Hint Check your answer.

REMINDER: After you have completed all 3 tables above, use the print button on your browser to print the tables and then bring them to lab.  You will need this information to complete the next section of the Prelab and you will also need it during 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.