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Teaching Data Analysis and Presentation– ABLE Poster - D. Hougen-Eitzman - Carleton College

Instructor's Note: Students will be required to plot the data from the Bradford Asssy into a standard curve, fit a line to the data, and use the equation for the line. These skills were introduced in the first lab computer session. LAB 2: Introduction to Lab Techniques I. INTRODUCTION (λ) absorbed by a substance. It In this , you will be introduced does this by measuring the intensity of light (or reintroduced) to some basic techniques after it has passed through the substance. of modern - , The essential components of the instrument and micropipetting. In are shown below in Figure 2.2. today’s lab we will practice the general use We make use of the phenomenon that the of the compound light . In absorption of light at a given wavelength is addition, we will conduct an that related to the concentration of the absorbing will introduce/reinforce the use of chemical. For instance, if I0 is the intensity spectrophotometers and micropipettors, as of the incident light (the light entering the well as exercise your computer skills. sample) and I is the intensity of the transmitted light (the light leaving the II. PROTEIN EXPERIMENT sample), then the absorbance is the relative In this lab exercise, we will practice two amount of light that is absorbed by the important and use lab techniques: using a sample: spectrophotometer and micropipetting. In A = log I0 /I the exercise, you will use these methods for a common type of assay in - Thus, if the intensity of light coming out of determining the protein concentration of an the sample is the same as the light going in, unknown sample. then no light has been absorbed and A = log (1) = 0. On the other hand, if the The determination of protein concentration absorbance is high, this means that very is frequently required in biochemical work. little light passed through the solution. In For instance, if we are comparing the the visible , you can think of water activity of 2 different enzyme preparations, as having an absorbance close to 0, while and find one to display substantially more milk has a large absorbance. Note that the activity than other, we really can't conclude relationship between absorbance and anything until we have ascertained how proportion of light transmitted is much of the each enzyme is present logarithmic, so a 10 times reduction in (remember that enzymes are proteins). transmitted light results in an increase in A. Spectrophotometry absorbance (A) of only 1. Spectrophotometric techniques are The conversion of absorbance to techniques based on the differential concentration of absorbing substance is absorption of light by different chemicals. straightforward: These techniques serve a wide array of A = ε c Beer-Lambert Law functions in biology. They can be used to l determine the concentration of many where A is absorbance, ε is the extinction compounds, such as DNA and proteins, and coefficient (a property of the compound that they can be used to measure enzyme is doing the absorbing), c is the activity. concentration of the absorbing material, and The machine we'll be using is a l is the length of the light path, usually 1 cm. spectrophotometer. It measures This little equation is so important that it has absorbance, the amount of light of a given

2-1 Teaching Data Analysis and Presentation– ABLE Poster - D. Hougen-Eitzman - Carleton College been made into a law -- the Beer-Lambert Law or sometimes Beer's Law.

2-2 Teaching Data Analysis and Presentation– ABLE Poster - D. Hougen-Eitzman - Carleton College

B. Use of Micropipettors Setting volumes on the pipetmen: Throughout this term, you will be required To use the pipettors, first set the volume to to accurately and reproducibly. This pipet using the dial on the pipet. See the is not as easy as it sounds. In fact, next to table below for example settings on the failing to read and think about the lab pipettors. directions, pipetting mistakes are the greatest source of heartache in the laboratory. for the P1000: To facilitate this important task, we will be 1 0 0 using highly accurate and sophisticated (and 0 5 2 expensive) automatic pipettors. There are several brands available, but we will be 0 0 5 using the Pipetman. You will need to make use of 3 different micropipettors: equals equals equals 1000 µl or 500 µl or 250 µl or 1) the P1000, which measures 200 to 1000 1 ml 0.5 ml 0.25 ml µl (microliters); 2) the P200, which measures 20 to 200 µl; for the P200: 3) and the P20, which measures 1 to 20 µl. 2 0 0 0 8 5 Fill out the following chart: 0 5 0 1 µl = ____ml; measure it with a P____ equals equals equals 10 µl = ____ml; measure it with a P____ 200 µl or 85 µl or 50 µl or 100 µl = ____ml; measure it with a P____ 0.2 ml 0.085 ml 0.05 ml

1000 µl = ____ml; measure it with a P____ for the P20:

2 1 0 Figure 2.3. All of 0 0 5 the micropipettors operate in the 0 5 5 same fashion. To choose the equals equals equals volume, hold the 20 µl or 10.5 µl or 5.5 µl or micropipettor 0.02 ml 0.0105 ml 0.0055 ml body in one hand and turn the volume adjustment knob until the correct volume shows on the digital indicator. The volumes are read from the top down.

2-3 Teaching Data Analysis and Presentation– ABLE Poster - D. Hougen-Eitzman - Carleton College

Using the pipetmen: Rules for pipetting

After you have dialed in the appropriate It is always good to have a list of rules. volume, attach a disposable tip to the shaft. Here is a list of rules for pipetting: The P1000 uses the large tips (usually blue or clear) and the P200 and P20 use the ⇒ Never rotate the volume adjuster smaller size (usually yellow). Next, depress beyond the upper or lower range of the plunger to the first stop. This part of the the pipettor. stroke is the calibrated volume displayed on the dial. Immerse the tip into the sample ⇒ Never force the volume adjuster. If liquid to a depth of several mm. Allow the force is required, you are doing plunger to return slowly to the up position. something wrong or the pipettor is Never let it snap up. (Why?) Withdraw the broken. In either case, see you tip from the liquid and remove any adhering instructor. In general, in liquid by touching to the inside of the tube as well as life, if force is required, holding the sample liquid. back off and think about it.

To dispense the sample, place the tip end ⇒ Never use the pipettor without a tip against the side wall of the receiving tube (No duh!). and depress the plunger slowly to the first stop. Wait a second and then depress the ⇒ Never lay down the pipettor with plunger to the second stop. With the liquid in the tip. plunger depressed, remove the tip from the tube, allow the plunger to return to the top ⇒ Never let the plunger snap back after position and discard the tip. withdrawing or ejecting sample.

The most common mistake in pipetting is using the second plunger stop to fill the ⇒ Never immerse the barrel into pipet – don't do this! :You should use the solution. first stop for filling the pipet, and the second stop for emptying it. Follow these rules throughout your life and you will be a great success in any molecular or biochemistry lab.

2-4 Teaching Data Analysis and Presentation– ABLE Poster - D. Hougen-Eitzman - Carleton College

C. Making a Standard Curve Instructor's Note: Students will be required The Bradford Assay to plot the data from the Bradford asssy into The Bradford assay is, more or less, the a standard curve, fit a line to the data, and gold standard of protein concentration use the equation for the line. These skills determination. It is quick and can detect were introduced in the first lab computer protein concentrations as low as 1µg/ml. session. The assay takes advantage of the fact that the color of the dye Coomassie Brilliant For this lab, we'll determine protein Blue G-250 changes when it is bound to concentration of an unknown sample using a proteins. This color change alters the standard curve. We will first measure the amount of light absorbed by the solution, absorbance of several samples of known which in turn can be measured with the protein concentration. We can then draw a spectrophotometer. The Coomassie blue graph of the relationship between binds primarily to basic and aromatic amino absorbance and protein concentration. Once acid residues, especially arginine. This we have this standard curve, we can specificity does introduce the problem of measure the absorbance of an unknown varying sensitivity, depending on the amino sample and read its corresponding protein acid composition of the proteins, but this is concentration from the graph. Without an compensated for by its ability to detect such accurate standard curve, well, there is just small amounts of protein. (Under what not much point in going on. circumstances would the specificity of the Coomassie blue binding a potential Preparation of the Standard Curve problem?) The Bradford reaction comes in We will construct a standard curve 2 forms, one which can detect 200 µg/ml to according to the table below. In light of the 1200 µg/ml, and a souped up version that discussion above, the standard curve must can detect 1µg/ml. We will use the less be linear. If it is not linear, do it again. sensitive version. However, before you redo the curve, graph it and show it to your lab instructor or teaching assistant in order to confirm that you need to do it again. Sometimes you are too hard on yourselves, although sometimes you give yourself too much slack, also.

We will do each tube in duplicate. This is always a good idea. (Why?) In our case, the duplicates must be within 10 % of the average of the two readings. (The acceptable difference varies with the nature of the experiment, the type of equipment, and the sophistication of the experimenter.)

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Directions for using the Spectrophotometer: 1. Turn on the spectrophotometer (the You will be reading all your samples in a switch is on the back of the machine (see illustration below). To transfer next to the power cord). When you turn your samples to the cuvette, pour from your on the Spectronic 20 Genesys instrument sample tube directly into the cuvette. Each ("spec 20"), it performs its automatic time you put the cuvette into the spec, you power-on sequence (check to be sure should wipe the outside of the cuvette with a that the cell holder is empty and its Kimwipe (a lab tissue). Any crud on the cover is closed before turning on the outside of your cuvette will give you an instrument). The self-check sequence erroneous reading and tend to build up takes about 2 minutes to complete; do inside the spec (a bad thing). After you take not interrupt it during this sequence. a reading, we recommend that you pour your Allow the instrument to warm up for sample back into the tube it came from, just about 30 minutes before you use it. in case you need to take a second reading later. After you get most of the sample back 2. The machine should be in "Absorbance in its original tube, gently tap the cuvette mode" – i.e., the display should show upside down on a Kimwipe to get as much some numbers followed by an "A". If fluid out as possible. Rinse out the cuvette not, press 'A/T/C' to select the with water from a squirt between absorbance mode. samples; again, tap the cuvette on a 3. Press nm or nm ' to set the Kimwipe to remove extra fluid. You do not wavelength to 595 nm. (The Coomassie need to dry the cuvette. blue absorbs light in the yellow portion All absorption measurements need to be of the spectrum, around 595 nm, leaving made relative to your blank (i.e., tube #1). only the blue-purple color that you see.) This solution contains all of the components Note: holding either key will cause the of the experimental tube, except the wavelength to change more rapidly than compound being measured. You can check pressing many times. your blank using tube 2, which should also 4. Next, set up your standard curve tubes give a reading of zero absorbance. Save (#1-12), including the five minute tubes 1 and 2 to re-blank the spec for parts B incubation, according to the table on the and C below. Think carefully about why next page. you are using tubes 1 and 2 to blank the 5. After the machine is warmed up, you spectrophotometer (why not just use need to blank the spectrophotometer. water?). Check with your TA if you're not To do this, pour the fluid from tube #1 sure. (see the table on the next page) into the Follow the protocol below. You should cuvette (see illustration below), wipe the display confidence. By the way, BSA cuvette with a kimwipe, and place the stands for bovine serum albumin, a blood cuvette into the spec 20, aligning the protein from cows. You have a very similar mark on the cuvette with the mark on the protein in your blood. It is essential for sample holder. Press the “0 maintaining proper osmotic conditions and ABS/100%T” button. The display is important in carrying fatty acids, which should show an absorbance of 0.000. are a common cellular fuel.

Spectrophotometer cuvette

2-6 Teaching Data Analysis and Presentation– ABLE Poster - D. Hougen-Eitzman - Carleton College

The Standard Curve

Note: All volumes are in ml Tube #: 1 2 3 4 5 6 7 8 9 10 11 12 Additions BSA (1.0 mg/ml) - - 0.02 0.02 0.04 0.04 0.06 0.06 0.08 0.08 0.1 0.1

Water 0.1 0.1 0.08 0.08 0.06 0.06 0.04 0.04 0.02 0.02 - - Mix each solution by gently shaking the tube, then add the Bradford Reagent. Bradford Reagent 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

Cover the tube with a small square of parafilm, invert several times to mix and then let stand at room temperature for at least 5 min. Insert tube #1 into the spec and zero the machine as described on the previous page. Then measure the absorbance at 595 nm for each of the other tubes and record these values in the table below. It is good form to re-blank the machine a couple of times (with tube #1) during these your runs.

Tube #: 1 2 3 4 5 6 7 8 9 10 11 12

A595 (Reading from the Spec 20)

Now, calculate how many micrograms of the standard protein were present in 0.1 ml of sample (that is, before you added the Bradford Reagent volume) and record that number below. (Hint: you can do this before coming to lab.)

Tube #: 1 2 3 4 5 6 7 8 9 10 11 12

µg of protein added:

Graphing a Standard Curve (don't average them) so you can tell if one of Using the graph paper included at the end the values is abnormal. Your graph should of this lab, plot the absorbance readings on produce a straight line. Fit a line to the the ordinate of a graph (y-axis) and the curve and record the equation for the best-fit line. amount of protein in µg on the abscissa (x- axis). Now show this graph to your lab With this standard curve, you can instructor or teaching assistant in order to determine the protein concentration of an confirm that your curve is linear. If it is not, unknown sample by recording its the most likely cause is pipetting errors. absorbance in the Bradford Assay, and then reading the corresponding amount of protein Now enter your data into a Microsoft off of the standard curve. Excel spreadsheet using a computer in the adjacent computer lab (Hulings 106) – enter the absorbance for each tube separately

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D. Determining the concentration of up and down. When you add the last protein in an unknown sample component (in this case the 30µl of unknown) to your microfuge tube, expel the fluid into the solution by putting the tip Preparation of the Unknown Sample of directly into the solution (rather than on the Bovine Serum Albumin (BSA) side of the microfuge tube above the solution). Then suck some of the solution It is essential that the absorbance for the back up into the pipet tip; expel this back unknown falls within in the parameters of into the microfuge tube and repeat several the standard curve. (Make sure that you times. If you use this technique, you must understand why this is so.) But, because it be careful not to get fluid up into the is an unknown, we don’t know for sure pipettor, especially when the volume whether this will be the case. One way measured is close to the capacity of the around this difficulty is to test the unknown pipettor! It is very easy to suck up too and if it is off of the standard curve, do it much fluid or air bubbles, which will cause again at a lower or higher concentration, fluid to rise into the barrel of the pipettor-- depending on which end of the standard this is very bad, since the pipettor then will curve the unknown fell off. Another way is not work properly. Watch closely when you to dilute the sample before we run the test. pipet small volumes. This is the approach we will take. Make sure you label your microfuge tube. You need to make a 1:10 and a 1:100 1 You will use 0.1 ml of this (in tubes 15 and dilution of your unknown. Once you make 16) for the Bradford assay below. up these dilutions, in smaller tubes, you can take samples from these to use in the 1:100 Dilution: Bradford assay. Add 0.297 ml of water to a clean, labeled 1:10 Dilution: microfuge tube. To this, add 3 µl of your unknown. Alternatively, you can make a Into a microfuge tube (these are small, 1:10 dilution of your 1:10 dilution. Mix , colored tubes found in a jar or carefully (see note above). beaker at your bench), pipette 0.27 ml of water. To this, add 30 µl of your unknown. You will use 0.1 ml of this (in tubes 17 and 18) for the Bradford assay below. Mix carefully--one common way in which biologists mix small volumes is by pipetting

1 A note on dilutions: Conventions for expressing dilutions vary from lab to lab, and sometimes from situation to situation. Usually, a 1:10 dilution means a "10-fold" or a "10x" dilution. When people write "1:10 dilution," they tend to mean 1 part of stuff you're diluting in ten parts TOTAL. You're making something ten times weaker than it started out. A more correct way of stating this as a true ratio is to call it a 1:9 dilution (one part stuff to nine parts buffer), and some people do this. One common exception to this is circumstances which require a large dilution (1:200, or 1:500), and the actual amount of stuff isn't critical. In these cases, people will actually use 200 parts of buffer, add one part of stuff, and disregard the extra part. The best thing to do is to ask for clarification when you’re in a new situation.

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Teaching Data Analysis and Presentation– ABLE Poster - D. Hougen-Eitzman - Carleton College

Determination of the Protein Concentration of an Unknown Sample of BSA Prepare a Bradford Assay according to the table below. Note: All volumes are in ml Tube #: 13 14 15 16 17 18 Additions Unknown (undiluted) 0.1 0.1 - - - - 1:10 Dilution of Unknown - - 0.1 0.1 - - 1:100 Dilution of Unknown - - - - 0.1 0.1 Mix the solutions by gently shaking the tube, then add the Bradford Reagent. Bradford reagent 5.0 5.0 5.0 5.0 5.0 5.0

Cover the tubes with parafilm, invert several times to mix and then let stand at room temperature for at least 5 min. Rezero your machine with tube #1. For each of tubes 13-18, pour the contents into a cuvette and measure percent absorbance at 595 nm. Record your readings in the table below.

Tube #: 13 14 15 16 17 18

A595 (Reading from the Spec 20)

Now, using the absorbance values of the unknown that you have just recorded and the equation for standard curve from above, determine how much protein was present in 0.1 ml (100 µl) in each of the experimental tubes (how much protein sample was added to each tube?) . Enter these values into the table below.

Tube #: 13 14 15 16 17 18 µg protein in 0.1 ml sample: (From Standard Curve)

Now calculate the concentration of your undiluted unknown in terms of mg/ml. Here is one way to do this: For each of the three dilutions, average the values for the duplicate samples. Then convert these averages from µg to mg by dividing by 1000 (which is the number of µg per mg). This will give you values in terms of mg per 0.1 ml, so to get to mg/ml, you’ll need to divide by 0.1. Finally, for the 1:10 dilution, multiply the resulting value by 10, and for the 1:100 dilution, multiply the resulting value by 100. These results will give you estimates of the concentration of your undiluted unknown in mg/ml. Did all three dilutions of your unknown yield the same final value of protein concentration? If not, why not? What values do you trust most? Why?

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III. LABORATORY WRITE-UP

Include only the following items in this week’s lab write-up. You are not responsible for turning in answers to the questions scattered throughout the manual. And this is not a group assignment; you should turn in your own work.

1) A copy of your sketch of an organism from the pondwater sample. Make sure to include the scale reference and a note of the magnification.

2) The BSA standard curve, including all components of a good graph (see Knisely's A Student Handbook for Writing in Biology, 2nd ed. for details). This should include a caption below your figure and clearly labeled axes.

3) A detailed calculation of the protein concentration (in mg/ml) of the unknown solution of BSA; include a calculation for each dilution.

4) State which dilution you have the most confidence in, and why.

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