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Bioc 111 honor code

Resources

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

Bioc 111 Day 6

Conduct a biuret protein assay

Spectrophotometer video

Even if you have already used a Spectrawave visible light spectrophotometer, reviewing the video should help you with today's work, as it provides valuable information on the protein assay itseslf.

Background

The biuret assay can provide a quantitative estimate of the concentration of protein so that we might analyze experimental results or optimize an experiment. Recall that biuret reagent changes color with intensity proportional to the concentration of protein in a sample (within limits). To estimate the protein concentration in a sample for which the concentration is not known we need to use standards for comparison. Standards are samples containing known amounts of protein. When we mix color reagent with the standards we obtain a range of color intensities to which to compare the unknowns.

One could estimate protein concentration of unknowns by comparing each unknown with the set of standards, but that method has obvious drawbacks. It relies on our judgment and of course there is what to do when the color change of an unknown falls between the color changes of two standards. Last time we introduced you to a device called a spectrophotometer, which converts color change to a quantity called an absorbance value. By measuring absorbance values corresponding to a set of protein standards we can plot a standard curve of absorbance versus amount of protein. Absorbance and amount of protein are continuous variables, so we should add a trend line that relates absorbance to amount over the entire usable range of the assay.

We can estimate the amount of protein in an unknown from its absorbance by reading the corresponding amount from the standard curve. Concentration of the unknown is simply the estimated amount divided by the volume of sample that was added to the tube.

Preparation

You will need to plan your standard curve ahead of time.

Experimental overview

Today you will start by conducting a protein assay. We will have you prepare the standard curve in your notebook, in class, and use it to estimate protein concentrations for two unknowns. We will then have you use the information to accomplish the kinds of objectives that are a part of many laboratory protocols.

• Prepare a set of protein standards, add color reagent, determine absorbances
• Prepare unknowns for assay, add color reagent, and measure absorbances
• Plot a standard curve in your notebook and add a trend line
• Estimate concentrations for your unknowns
• Calculate how to dilute your unknowns using two different approaches
• Estimate fraction yields for your two unknowns

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

A) Prepare protein standards

1. Referring to the table in step 3 below, calculate the volume of 20 mg/ml protein standard to go into each assay tube.
2. We want to normalize the volumes to 1 ml per tube before adding color reagent. Otherwise, variable volumes will affect color intensity and distort the results. Determine the volume of water to add to each tube so that the starting volume of every tube is 1 ml. Bring these numbers to lab with you
3. Under the heading "Protein standards," set up a table in your notebook as in the example below. Fill in the two volume columns with the values that you calculated prior to lab. Leave the absorbance column blank for now.

4. Obtain 11 13/100 mm culture tubes and place them in a peg rack, open ends up.
5. 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. Label the remaining 10 tubes 1 through 10.
6. 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.

B) Add color reagent, incubate, read and record the absorbance values

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.
2. While your tubes are incubating at the bench (10 minutes), turn on and calibrate your spectrophotometer as you learned last time. The reference tube does not require the 10 minute incubation..
3. 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.
4. Keep your reference tube to re-check your calibration when you read your unknowns.

C) 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 four columns. Label columns 1-4 "tube number," "volume of unknown," "volume of water," and "absorbance @ 540 nm."
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, and read your absorbances, recording them in column 4 of your unknowns table.
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 before you leave for the day.

D) Clean up

Before starting on the calculations (parts E-G), please clean all equipment and supplies and straighten up your bench area as you did for Day 5.

NOTE: Please start parts E-G on a new notebook page. We must be able to give copies of your lab notes to your TA for spot-checking and your work in parts D-F to an instructor for grading.

E) Prepare your standard curve and estimate protein concentrations

Your protein standard curve will serve as a tool for estimating protein concentrations in lab. Therefore you want it to be fairly large – it should take up most of the width of the page and be well-proportioned as suggested in the graphing tutorial that you completed earlier in the course. Choose informative labels for the axes as you learned previously. Plot your standard curve data and then include a best fit trend line. The relationship may be linear or somewhat curvilinear. Use your best judgment to fit your trend line, and do remember that it is not good practice to extrapolate, either toward or away from the origin.

From the absorbances for your unknowns estimate each protein concentration. Remember that concentration of the unknown is the amount of protein divided by the volume of sample used, not total volume in the assay tube. By convention we nearly always report protein concentrations as milligrams/milliliter (mg/ml). For each unknown also remember to use the single absorbance value that falls within the most linear part of the standard curve.

F) Plan to make dilutions

Show all work. 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.

1. Your first problem is to determine how to dilute 150 µl of each of your two unknowns 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.
2. Your second problem is to determine how to dilute the 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.

G) 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. We usually start by homogenizing the tissue, then we separate the homogenate into components, often employing a method called differential centrifugation. Centrifugation yields a solid component (the pellet) that we resuspend in a volume of liquid. It also yields a liquid component, the supernatant, that we process further. 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 each of your two unknowns was a sample from the fraction with corresponding label (e.g., unknown A was a sample from fraction A, etc.).

For each of the two unknowns that you selected, use the volume in the table to determine the total protein in the fraction. Enter the results in your notebook, showing how you did the calculation.

Homework due

Today you turn in your final draft of materials and methods for DNA labs 1-4. Please use a paper clip to attach your graded draft to the back of your revised methods section. There will be no homework assignments following day 6. Enjoy!

We would like to thank New England Biolabs for their generous support of this laboratory course