Summer Research Program for Science Teachers

Rosa Jimenez

Washington Irving High School, Manhattan

1999

Teaching Students How to Read the Protein Concentration in a Sample

MATERIALS:

8 cuvettes

sample (i.e. extract from E.coli)

pipette

bovine serum albumin (BSA)

water

two 20-30 ml test tubes

spectrophotometer

[Teaching Standard D- Make accessible science tools]

PROCEDURE

· Dilute Bio Rad 1:4. To do this, take, let's say, 1 ml of Bio Rad and mix it with 4 ml of water.

· Prepare BSA 1mg/ml or 1ug/l. Take 1g of powder BSA and mix it with 1 l of water or buffer.

· Take five cuvettes and label 1-5.

· Cuvette number 1 2 3 4 5

BSA (1ug/ul) 2ul 4ul 6ul 8ul 10ul

Bio Rad (1:4) 998ul 996ul 994ul 992ul 990ul

Record reading at X nm using spectrophotometer

· Plot a curve: adsorbance (nm) vs. BSA (ug) or vice versa [9-12 Content Standard A- Use mathematics to improve communication]

· Then, choose between 2ul to 10 ul of your unknown sample and the appropriate amount of Bio Rad (1:4) using the scale shown above. For instance if you take 4ul of the sample, you need to add 996 ul of BioRad to make a final volume of 1,000 ul in a cuvette. Once you have done this, take a reading of the mixture in the spectrophotometer at the same absorbance as above. Plot the reading of the unknown into the curve you plotted and this will tell you the approximate amount (ug) of the unknown sample. [Content Standard Unifying Concepts- Change, constancy, and measurement] Repeat this two more times to make sure you got an accurate reading. Once you know the ug amount, you divide that by the volume of the unknown that you chose and this will tell you the protein concentration of the unknown sample.

· This protocol is modified based on the Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [Teaching Standard D- Make accessible science media]

DEVELOPMENT

In this exercise, students will work in groups of two. The students will not be provided with all the calculations that they need because I want them to discuss the possibilities among themselves. This will help them to reason out a logical final volume for the BSA and the Bio Rad based on the size of the test tubes and the cuvettes that are available to them.

My role will be that of a facilitator. [Teaching Standard A- Select strategies to develop student understanding] I will make sure that they have the materials and equipment readily available. At no point should I do any of the steps for the students. The idea of the exercise is that they do all the steps on their own as carefully as possible. I expect many mistakes to be made the first time that they do this lab. However, this lab should be repeated for those who had difficulty, whereby the students who understood the exercise well provide help and guidance for those who had difficulties.

To have individual accountability, all students will submit a written laboratory report, which should include introduction, methods, results (showing all tables and graphs), and discussion/conclusion.