The purpose of this lab is for you to
1.
Gain hands-on practice with quantifications of proteins
2.
Reinforce your knowledge of spectrophotometry
3.
Practice using appropriate and efficient lab procedures to answer research questions.
4.
Read the background in section D. below.
5.
Review the use of the Spec 20 here: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html
and write a step by step protocol for using the Spec 20 in your lab notebook.
6.
Review the Beer-Lambert Law on the same website, and be prepared to use the
Beer-Lambert Equation in class to determine an absorption coefficient and determine the concentration of an unknown sample.
7.
Work in a group of two and follow the instructions under E.
One of the most common procedures used in a biochemistry laboratory is the quantification of proteins. Protein concentrations must be carefully controlled for a wide variety of reasons. In the research or medical laboratory, the amount of protein used in each experiment must be carefully measured and recorded. If a researcher is running a protein gel to visualize the molecular weight of a protein, she needs to be sure to load enough protein to detect, but not so much that she overloads the gel. If a graduate student is using an enzyme to catalyze a chemical reaction, he needs to add enough enzyme so that his reaction proceeds optimally – and he may need to test a variety of different concentrations to determine what that optimum is! A scientist working for a diagnostics company, developing new antibody-based diagnostic tests needs to carefully determine the concentration of antibody that is used in your new products. Many of the
“latest and greatest” new drugs are protein-based. Thus, precise methods must be in place to accurately measure protein concentration during the manufacturing process. In short – protein concentration determination is a ubiquitous and indispensible tool in any modern biology lab.
Proteins are often valuable – difficult to purify and isolate – and present in only very small amounts. This is one of the reasons that new, antibody-based therapeutics are so incredibly expensive. It’s usually not practical to simply weigh your protein sample on a balance. Often, the researcher has far too little material to actually weigh, and usually the protein is in some buffered solution. The quantification of protein depends on comparing your UNKNOWN protein solution to a STANDARD protein solution. Protein standards are usually prepared by weighing out an inexpensive and abundant protein in dry form, and making a standard solution that is of a known concentration. Bovine serum albumin (BSA) is one such abundant protein, and is commonly used as a protein reference standard. Bovine IgG also may be used in many cases.
Quantification of proteins, Sheri Miraglia@CCSF 1

In today’s lab, you will be creating your own protein standard curve, and determining the concentration of an unknown protein sample. We will be using a protein assay called a
“Bradford Assay” that uses Coomassie Brilliant Blue Dye. Coomassie Blue binds to the proteins in solution, and can be detected spectrophotometrically. When the dye is free, it has an absorption maximum of 465 nm, but when it binds to protein, the absorption maximum shifts to 595 nm ( Figure 1 ).
Figure 1.
Spectra of Coomassie Blue unbound (bottom) and bound (top) to protein.
When preparing a standard curve, one of the first considerations is the set of standards.
What concentration range is appropriate? The range is different depending on the type and sensitivity of the assay. For the Bradford Assay, the typical linear range is between
5-100 ug protein per assay tube. In Figure 2 , a typical standard curve graph is shown.
Note that the assay is linear from 0-1000 ug (for this hypothetical assay), but that as the concentration increases, linearity is lost and the absorbance begins to flatten out.
Standard curves are reliable ONLY within the linear range of the curve. If one had an unknown sample that had an assay absorbance roughly where the question mark is, he or she could use this curve to estimate the concentration of their unknown, using the
Beer-Lambert Equation.
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Figure 2.
Using a standard curve to determine the concentration of protein in an unknown sample.
Preparation of Standards:
Working in groups of two, select six concentrations to prepare from a 1 mg/mL solution of protein standard. In your lab notebooks, you will want to create a table with rows for each standard indicating the total protein in each tube, the amount of 1 mg/mL standard protein solution, and the amount of water used to make each standard. How many standards should you prepare for each point on your curve? Is a single tube enough?
Should you do the assay in duplicate? Triplicate? Discuss this with your lab partner and be prepared to justify your decision. When preparing your standard curve, think carefully about the predicted linear range for the Bradford Assay. Before preparing your samples, have your instructor review and approve your selection of assay standards.
Note: Each assay tube will need 100 ul of standard.
Preparation of Unknown:
Each group of two will receive their unknown protein tube upon receiving approval from the instructor for their standard curve plan. There is no guarantee that your unknown will fall within the range of the Bradford assay (although it won’t be TOO dilute to test).
Think about how much volume is in the tube. How many tests of this sample can you do? What is a logical way to proceed, given that you don’t have any initial information about the concentration of the unknown?
Assay:
You should have reviewed use of the Spec 20 before class. Warm up the spectrophotometer (Spec 20) if it has not already been turned on. For each sample, add
5 mL of Bradford dye reagent and incubate for 5 minutes in a glass cuvette prior to r eading the absorbance on the Spec 20. You will need to generate a “blank” for use in the Spec 20 (hint: this may already be one of your standards). Measure the absorbance for each standard (and replicates) and write it down in a table in your laboratory
Quantification of proteins, Sheri Miraglia@CCSF 3

notebook. Measure the absorbance of your unknown sample(s). If necessary, dilute your unknown and measure it again until your unknown absorbance falls within the range of your standard curve.
Create a graph with protein (in micrograms or micrograms/mL) on the X-axis and absorbance on the Y-axis. For each standard, calculate the standard deviation between your replicates and use this to generate error bars at each point (your instructor can help you with this as needed). Keeping the error bars in mind, determine where the best linear “fit” is within your standards and draw a straight line. Once you have a straight line, you can use the Beer-Lambert Law to calculate the absorbance coefficient for your assay, and the concentration of your unknown sample. The group that comes the closest to identifying the actual concentration of their unknown will win a small prize!
1.
What is the primary advantage of preparing standard curves and unknown samples in triplicate versus in duplicate? What would be a disadvantage?
2.
What is the problem with an unknown sample that has an absorbance that is not within the linear range of the standard curve?
3.
What is the value of error bars when preparing a standard curve?
4.
Coomassie Blue binds to hydrophobic pockets within the proteins being detected, and more specifically with arginine residues and less so with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues. What limitations would this cause when using this assay routinely for unknown proteins?
5.
The Biuret test is another protein assay method where copper ions bind to the peptide bond in the presence of strong base. What advantages might this method have over the method we used in class today?
References
Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.
1976.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html
, accessed 9/14/2011
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