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 wavelength (λ) absorbed by a substance. It In this laboratory, 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 biology- microscopy, The essential components of the instrument spectrophotometry 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 microscope. In absorption of light at a given wavelength is addition, we will conduct an experiment 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 ASSAY 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 biochemistry - 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 spectrum, 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 pipette 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 laboratories 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.