• Day 1
• Day 2
• Day 3
• Day 4
• Day 5
• Day 6

Resources

• *LAB SAFETY**
• Laboratory notebook
• Graphing tutorial
• Molecular biol tips
• Writing assignment
• Bios 211 web site
• Searching literature

Other links

• Bios 111 honor code
• Graphing quiz (rice only)

Bios 111 Day 5 Assignments

Introduction

An assay for protein concentration is an essential tool for biologists and biochemists. Many of the principles apply to assays for other substances as well.

Background

We very often need a quantitative estimate of the concentration of a substance in order to optimize an experiment. A colorimetric assay for protein concentration employs a reagent that changes color when mixed with protein. It requires the use of standards, which are tubes containing different amounts of the substance to be measured, each in solution in the same volume. We mix color reagent with the standards to produce color changes that are proportional to the amounts of substance in the tubes.

We use a device called a spectrophotometer to convert color change to a quantity called an absorbance value. We also prepare tubes containing different volumes of the unknowns, which are samples of unknown concentration, add color reagent to the unknowns, and read their absorbance values. You will conduct the analysis as part of your homework assignment.

We will employ a method called the biruet protein assay. The biuret assay is more than fifty years old, but we are using it because unlike many assays you can prepare the color reagent yourself from inexpensive inorganic salts. The ability to plan and prepare complex solutions is another essential skill that is usually taken for granted if you work in a biology or biochemistry laboratory.

Experimental overview

Today you will learn to prepare a complex solution (biuret reagent) and conduct a colorimetric assay. You will plot your data and conduct some calculations as a homework assignment. Each of you will work independently, recording all procedures and data.

• Prepare biuret reagent for a protein assay
• Prepare a set of protein standards
• Prepare unknowns for assay
• Add color reagent to standards and unknowns
• Calibrate a spectrophotometer
• Read and record absorbance values

***You must wear eye protection throughout this laboratory session***

A) Prepare your spectrophotometer

A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light. The instruments are arranged so that liquid in a glass container (called a cuvette) can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device. The signal changes as the amount of light absorbed by the liquid changes.

The spectrophotometers that we use require about 15 minutes warm-up time. The protocol for preparing the instrument is probably the easiest of all of them.

1. Open the lid to the sample chamber.
2. If someone has left a tube in the chamber (this is a "no-no"!) then remove it.
3. Close the lid and turn on the spectrophotometer using the knob on the left front of the instrument.
4. Check the wavelength (in nanometers) and use the adjacent knob to set it to 540 nm if it isn't there already.

B) Prepare biuret color reagent

Biuret reagent consists of 1.8 gm sodium potassium tartrate (f.w. 282.22), 0.6 gm copper sulfate x 5 H2O (f.w. 249.68), and 1 gm potassium iodide (f.w. 166.0), all dissolved in order in 80 ml 0.2 M NaOH (f.w. 40.0), then brought to a final volume of 200 ml. This is one of several ways to describe a solution formula.

***You must wear eye protection during the remaining procedures***

1. Place a piece of weigh paper on the left pan of your balance. Tare the balance by moving the small slider until the pointer is centered.
2. Move the slider further to add 0.64 g to the reading. You cannot be extremely accurate with this balance, but accuracy to precisely 0.64 g is not essential.
3. Weigh out 0.64 g NaOH by adding NaOH pellets until the pointer is again centered.
4. Add the NaOH to 70 ml deionized water and stir using a stir bar and plate.
5. When the NaOH is completely dissolved, q.s to 80 ml using the 100 ml cylinder. "Q.s." means "bring the solution to final volume." In this case pour the solution into a graduated cylinder of appropriate size and add deionized water to the 80 ml mark. You now have 80 ml of 0.2M NaOH.
6. Return the solution to the flask and stir to mix.
7. Weigh out, add, and completely dissolve 1.8 g sodium potassium tartrate. If any material is caught in the neck of the flask, use a Pasteur pipet to take up solution and rinse the inside surface.
8. Weigh out, add, and completely dissolve 0.6 g copper sulfate.
9. Weigh out, add, and completely dissolve 1.0 g potassium iodide.
10. Q.s. to 200 ml using the 250 ml cylinder.
11. With the completed reagent at full volume, return it to the flask and stir to mix completely. Your reagent should be clear blue.

Notice how each solution was prepared. We started with a volume of water less than the volume needed for the final solution. That way, the concentrations are right when we finish. If you started with a full 80 ml distilled water and then added 0.64 g NaOH, the volume would exceed 80 ml and you would not have a 0.2 M solution. Likewise, the concentrations of the remaining reagents would be too low if we brought the solution to 200 ml before we added the chemicals.

C) Prepare protein standards

In this part you will practice using variable volume micropipettors and work with amounts, volumes, concentrations, and associated units of measurement.

1. Under the heading "Protein standards," set up a table in your notebook as in the example below. Leave the Absorbance column blank for now.

2. Obtain 11 13/100 mm culture tubes and place them in a peg rack, open ends up.
3. Label one tube "R" using a black Sharpie marker. Label near the top of the tube, so that the label does not obstruct the light passing through the color reagent later. Place the tube in a peg rack.
4. Label the remaining 10 tubes 1 through 10.
5. Use a variable volume pipettor to deliver 20 mg/ml BSA and water into your tubes, according to the information listed in your table. You will need two different pipettors, one to deliver volumes under 200 µl and one to deliver volumes from 200 µl to 1 ml (1000 µl). It is very easy to forget which tube you are on. Experienced researchers check off each item in the notebook line by line and/or set each finished tube back one row in the rack to avoid confusion.
6. Don't forget to record in your notebook that you completed preparation of the standards.

D) Add color reagent and calibrate a spectrophotometer

1. When you are ready, use a serological pipet to add 5 ml color reagent to your reference tube and to each of your standards. A TA or instructor can show you how to use the pipet aid to draw up and deliver solution.
2.  Let your tubes stand for 10 minutes.
3. While the color develops ask a TA or instructor to show you how to calibrate the spectrophotometer and how to read absorbance values.

E) Read and record the absorbance values

According to Beer's Law the concentration of a substance is directly proportional to absorbance. Do make sure that you read the absorbance scale on your spectrophotometer. The concentration is not proportional to transmittance.

1. In the order in which you added color reagent, read and record the absorbance (not transmittance) corresponding to each of your standards. For best results the time interval between adding color reagent and reading absorbance should be close to the same for each tube.
2. Keep your reference tube to re-check your calibration when you read your unknowns.
3. While your unknowns are standing (step --- below), you may empty the used biuret reagent in the sink, running water to dilute the reagent as it goes down. Save the unused reagent.

F) Prepare unknowns and read absorbance values

You will need to prepare unknowns for comparison with the standards. Of course you should record all of the information in your notebook as you go along.

1. Obtain two 15 ml plastic conical centrifuge tubes containing samples of unknown concentration. One should be labeled A, B, or C and the other should be labeled D, E, or F.
2. Label two tubes with the letter code for one of your unknowns, followed by 1 or 2 (e.g., A-1 and A-2). Label two more tubes for your second unknown.
3. In your notebook set up a table for your unknowns as you did for the standards, but with six columns. Label columns 1-6 "tube number," "volume of unknown," "volume of water," "absorbance @ 540 nm," "amount protein (mg)," and "protein concentration (mg/ml)," respectively.
4. As you prepare your unknowns, fill out columns 1-3. For each unknown, tube 1 should contain 0.1 ml (100µl) of sample. Plan to put 1.0 ml of sample into each tube 2.

We prepare two tubes for each unknown so that if one tube contains too much or too little protein to be measured, the other tube should give us a usable reading.

5. Pipet the tabled volume of unknown into each respective tube, followed by the tabled volume of water.
6. Add color reagent, incubate, check your spectrophotometer calibration, read and record your absorbances.
7. Check that at least one absorbance reading for each of your unknowns falls within the range of absorbance values of your standards. If your data are "good," then you have all of the information you need with which to complete the rest of the work on your own time.

Homework Assignments

Prepare all of the homework assignments in your laboratory notebook so that you have duplicates to turn in at the beginning of the next laboratory session. You are strongly advised to use the "survival manual" as a reference to help you complete the homework.

1. Prepare a protein standard curve

In your notebook, set up axes to plot absorbance versus amount of protein, each using a linear scale. Neatly sketch a best fit line to the data, taking care not to extrapolate the relationship. The theoretical relationship is linear, although with most assays there is curvature to the relationship at higher amounts of protein as the reagent approaches saturation. If the points clearly point to a linear or a smooth curvilinear relationship then you may interpolate (connect individual data points). If they are too scattered then you may have to use a best fit line.

2. Determine concentrations of your unknowns

The whole purpose of the assay was to obtain an estimate of the amount of protein per unit volume in the original sample. For each unknown identify the tube that gave you the most reliable reading, namely the absorbance value that is closest to the middle of the standard curve. In most cases the other tube will give you an absorbance too high or too low to be used with the standard curve.

Estimate the amount of protein in mg that corresponds to the absorbance, and from this estimate and the known volume of unknown that you placed in each tube you can estimate the concentration of protein in the original sample. We typically report a protein concentration in mg/ml.

3. Dilution problems

First, we will have you dilute a specific starting volume to a desired final concentration of protein. This is the kind of dilution that you would perform in order to make a working solution. Second, you will determine how to prepare each of your samples to a desired final volume and concentration.

Start with a known volume

Your first problem is to determine how to dilute 150 µl of each unknown to a final concentration of 1 mg/ml. You know v₁, you determined c₁ using your standard curve, and your desired final concentration of 1 mg/ml is c₂. In your notebook record the three known variables for diluting each of your unknowns. Calculate v2, showing all calculations in your notebook. Write down both v₂ and the volume to add to v₁.

Prepare a desired volume at a predetermined concentration

Your second problem is to determine how to dilute each unknown to obtain a final volume of 150 µl at a final concentration of 1.5 mg/ml. Again record the three known variables and determine the unknown variable for each unknown. Show all calculations.

4. Estimate fraction yields

A common approach to learning how something works is to take it apart. We apply that principle to living tissue when we conduct what we call a tissue fractionation. When we conduct a fractionation we want to be able to report how much of each component we have, usually in terms of the amount of protein recovered.

Hypothetical fraction volumes

The following table lists fractions and volumes obtained from a hypothetical fractionation of whole liver tissue. For the purpose of this exercise, assume that the labels on your unknowns correspond to the fractions listed in the table.

From the protein concentrations that you determined and the volumes in the table, determine the total protein in each of the liver fractions and enter the results in your notebook.

Reminder

The final version of the materials and methods section is due the last week of labs on your lab day.