Modern experimental biochemistry pdf free download

A valuable reference for instructors and students, it's particularly useful to instructors who prefer to use their own customized experiments. Part II, Experiments, offers optimum flexibility through 15 tested experiments designed to accommodate the capabilities of laboratories and students at most four-year schools. Alternate methods are suggested and labs may be divided into manageable hour segments. The book offers the latest safety and environmental precautions in each experiment to inform students and instructors of potential hazards and proper disposal of materials.

For anyone interested in science. Biology and other natural sciences. ISBN Your tags:. There is ample information on the human body, its genome, and the action of muscles, eyes, and the brain. The complete set deals with the natural world, treating the metabolism of bacteria, toxins, antibiotics, specialized compounds made by plants, photosynthesis, luminescence of fireflies, among many other topics. The manual's stand-alone experiments are effective in courses meeting only once a week, giving students a broad overview of the subject matter.

A more comprehensive set of experiments is also available and allows students to delve further into each of the topics presented. The Second Edition also features new and revised experiments, including a new experiment that involves cloning the barracuda LDH gene!

Students and professors will also find expanded problem sets in this edition. Tip boxes, located throughout the text, provide pointers to students on how to perform the experiment at hand, while Essential Information boxes highlight pertinent information that will help the student complete the experiment.

The second edition continues to include references and further readings at the end of each chapter. Important Notice: Media content referenced within the product description or the product text may not be available in the ebook version. An investigational approach based on fundamental scientific principles forms the basis of experimental biochemistry. This book is designed with a holistic overview, encompassing various topics in the practice of experimental biochemistry, so that the information provided is useful to both the student and the teaching community.

The authors have applied a concept-oriented objective style in presenting the information in the text, rather than simply providing the facts, thus making this book eminently readable and easy to use. The purpose of the authors is to bring out an experimental manual that caters to the needs of graduate and postgraduate students, and which can be used as a supplementary text for biochemistry and biology courses.

Biochemistry in the Lab Author : Benjamin F. You can write a book review and share your experiences. Other readers will always be interested in your opinion of the books you've read. Whether you've loved the book or not, if you give your honest and detailed thoughts then people will find new books that are right for them.

American Society of Plant Taxonomists. Toby Pennington. Academic Press. Nathan P. Colowick , Nathan P. Kaplan , Klaus Mosbach. Water that is purified only by ion exchange will be low in metal ion concentration, but may contain certain organics that are washed from the ion-exchange resin. These contaminants will increase the ultraviolet absorbance properties of water.

If sensitive ultraviolet absorbance measurements are to be made, distilled water is better than deionized. Solution Preparation. The concentrations for solutions used in biochemistry may be expressed in many different units.

In your biochemistry laboratory, the most common units will be: Molarity M ; concentration per liter of solution. Many solutions you use will be based on molarity. For practice, assume you require 1 liter of solution that is 0.

Add about mL of purified water and swirl to dissolve. Then add water so that the bottom of the meniscus is at the etched line on the flask. Stopper and mix well. The flask must be labeled with solution contents 0. In general, solid solutes should be weighed on weighing paper or plastic weighing boats, using an analytical or top-loading balance. Liquids are more conveniently dispensed by volumetric techniques; however, this assumes that the density is known.

If a small amount of a liquid is to be weighed, it should be added to a tared flask by means of a disposable Pasteur pipet with a latex bulb. The hazardous properties of all materials should be known before use and the proper safety precautions obeyed.

The storage conditions of reagents and solutions are especially critical. Although some will remain stable indefinitely at room temperature, it is good practice to store all solutions in a closed container. This inhibits bacterial growth and slows decomposition of the reagents. If these are aqueous solutions or others that will freeze, be sure there is room for expansion inside the container. Stored solutions must always have a label containing the name and concentration of the solution, the date prepared, and the name of the preparer.

Volumetric flasks, of course, have glass stoppers, but test tubes, Erlenmeyer flasks, bottles, and. Remember that hydrocarbon foil, a wax, is dissolved by solutions containing nonpolar organic solvents. Bottles of pure chemicals and reagents should also be properly stored. Many manufacturers now include the best storage conditions for a reagent on the label. Many biochemical reagents form hydrates by taking up moisture from the air.

If the water content of a reagent increases, the molecular weight and purity of the reagent change. However, if this reagent is stored in a moist refrigerator or freezer outside a desiccator, the moisture content may change to an unknown value. Practical biochemistry is highly reliant on analytical methods. Many analytical techniques must be mastered, but few are as important as the quantitative transfer of solutions.

Some type of pipet will almost always be used in liquid transfer. Since students may not be familiar with the many types of pipets and the proper techniques in pipetting, this instruction is included here. Filling a Pipet. The use of any pipet requires some means of drawing reagent into the pipet. Liquids should never be drawn into a pipet by mouth suction on the end of the pipet. Small latex bulbs are available for use with disposable pipets Figure 1AA.

For volumetric and graduated pipets, two types of bulbs are available. One type Figure lAB features a special conical fitting that accommodates common sizes of pipets. To use these, first place the pipet tip below the surface of the liquid.

Squeeze the bulb with your left hand if you are a right-handed pipettor and then hold it tightly to the end of the pipet. Slowly release the pressure on the bulb to allow liquid to rise to 2 or 3 ern above the top graduated mark.

Then, remove the bulb and quickly grasp the pipet with your index finger over the top end of the pipet. The level of solution in the pipet will fall slightly, but not below the top graduated mark. If it does fall too low, use the bulb to refill. Mechanical pipet fillers sometimes called safety pipet fillers, propipets, or pi-fillers are more convenient than latex bulbs Figure 1.

Equipped with a system of hand-operated valves, these fillers can be used for the complete transfer of a liquid. The use of a safety pipet filler is outlined in Figure 1. Never allow any solvent or solution to enter thepipet bulb. To avoid this, two things must be kept in mind: 1 always maintain careful control while using valve S to fill the pipet, and 2 never use valve S unless the pipet tip is.

Examples of pipets and pipet fillers. If the tip moves above the surface of the liquid, air will be sucked into the pipet and solution will be flushed into the bulb. Other pipet fillers are used in a similar fashion. Disposable Pasteur Pipets. Often it is necessary to perform a semiquantitative transfer of a small volume 1 to 10 mL ofliquid from one vessel to another. Since pouring is not efficient, a Pasteur pipet with a small latex bulb may be used Figure I. Pasteur pipets are available in two lengths 15 em and 23 em and hold about 2 mL of solution.

These are especially convenient for the transfer of nongraduated amounts to and from test tubes. If dilution is not a problem, rinsing the original vessel with a solvent will increase the transfer yield. Used disposable pipets should be discarded in special containers for broken glass. Calibrated Pipets. Although most quantitative transfers are now done with automatic pipetting devices, which are described later in the chapter, instructions will be.

How to use a Spectroline safety pipet filler. Using thumb and forefinger, press on valve A and squeeze bulb with other fingers to produce a vacuum for aspiration. Release valve A leaving bulb compressed. To deliver the last drop, maintain pressure on valve E, cover E inlet with middle finger, and squeeze the small bulb.

If a quantitative transfer of a specific and accurate volume of liquid is required, some form of calibrated pipet must be used. Volumetric pipets Figure 1AF are used for the delivery of liquids required in whole milliliter amounts 1, 2, 3, 4, 5, 10, 15,20,25, 50, and mL.

To use these pipets, draw liquid with a latex bulb or mechanical pipet filler to a level 2 to 3 em above the "fill line. Release liquid from the pipet until the bottom of the meniscus is directly on the fill line.

Transfer the pipet to the inside of the second container and release the liquid. Hold the pipet vertically, allow the solution to drain until the flow stops, and then wait an additional 5 to 10 seconds. Touch the tip of the pipet to the inside of the container to release the last drop from the outside of the tip.

Remove the pipet from the container. Some liquid may still remain in the tip. Most volumetric pipets are calibrated as "TD" to deliver , which means the intended volume is transferred without final blow-out, i. Fractional volumes of liquid are transferred with graduated pipets, which are available in two types-Mohr and serological.

Mohr pipets Figure lAG are available in long- or short-tip styles. Long-tip pipets are especially attractive for transfer to and from vessels with small openings.

Virtually all Mohr pipets are TD and are available in many sizes 0. The marked subdivisions are usually 0. Selection of the proper size of pipet is especially important. For instance, do not try to transfer 0. Use the smallest pipet that is practical. The use of a Mohr pipet is similar to that of a volumetric pipet.

Draw the liquid into the pipet with a pipet filler to a level about 2 em above the "0" mark. Lower the liquid level to the 0 mark. Remove the last drop from the tip by touching it to the inside of the glass container. Transfer the pipet to the receiving container and release the desired amount of solution. The solution should not be allowed to move below the last graduated mark on the pipet. Touch off the last drop.

Serological pipets Figure 1. Their use is identical to that of a Mohr pipet except that the last bit of solution remaining in the tip must be forced out into the receiving container with a rubber bulb. This final blow-out should be done after 15 to 20 seconds of draining.

Automatic Pipetting Systems. For most quantitative transfers, including many identical small-volume transfers, a mechanical microliter pipettor Eppendorf type is ideal. This allows accurate, precise, and rapid dispensing of fixed volumes from 1 to u. The pipet's push-button system can be operated with one hand, and it is fitted with detachable polypropylene tips Figure 1. Other useful information about pipetting is available at the Web site www.

The advantage of polypropylene tips is that the reagent film remaining in the pipet after delivery is much less than for glass tips. Mechanical pipettors are available in up to 25 different sizes. Newer models offer continuous volume adjustment, so a single model can be used for delivery of specific volumes within a certain range.

To use the pipettor, choose the proper size and place a polypropylene pipet tip firmly onto the cone as shown in Figure 1. Tips for pipets are. A How to use an adjustable pipetting device. B Set the digital micrometer to the desired volume using the adjustment knob. Attach a new disposable tip to the shaft of the pipet.

Press on firmly with a slight twisting motion. C Depress the plunger to the first positive stop, immerse the disposable tip into the sample liquid to a depth of 2 to 4 mm, and allow the pushbutton to return slowly to the up position and wait I to 2 seconds.

D To dispense sample, place the tip end against the side wall of the receiving vessel and depress the plunger slowly to the first stop. Wait 2 to 3 seconds, and then depress the plunger to the second stop to achieve final blow-out. Withdraw the device from the vessel carefully with the tip sliding along the inside wall of the vessel.

Allow the plunger to return to the up position. Discard the tip by depressing the tip ejector button. Photos courtesy of Rainin Instrument Company, Inc. Pipetman is a registered trademark of Gilson Medical Electronics. Exclusive license to Rainin Instrument Company, Inc.

Details of the operation of an adjustable pipet are. For rapid and accurate transfer of volumes greater than 5 mL, automatic repetitive dispensers are commercially available. These are particularly useful for the transfer of corrosive materials. The dispensers, which are available in several sizes, are simple to use. The volume of liquid to be dispensed is mechanically set; the syringe plunger is lifted for filling and pressed downward for dispensing.

Hold the receiving container under the spout while depressing the plunger. Touch off the last drop on the inside wall of the receiving container. Special procedures are required for cleaning glass pipets. Immediately after use, every pipet should be placed, tip up, in a vertical cylinder containing a dilute detergent solution less than 0.

The pipet must be completely covered with solution. This ensures that any reagent remaining in the pipet is forced out through the tip. If reagents are allowed to dry inside a pipet, the tips can easily become clogged and are very difficult to open.

After several pipets have accumulated in the detergent solution, the pipets should be transferred to a pipet rinser. Pipet rinsers continually cycle fresh water through the pipets.

Immediately after detergent wash, tap water may be used to rinse the pipets, but distilled water should be used for the final rinse. Pipets may then be dried in an oven. The purpose of each laboratory exercise in this book is to observe and measure characteristics of a biomolecule or a biological system.

The characteristic is often quantitative, a single number or a group of numbers. These measured characteristics may be the molecular weight of a protein, the pH of a buffer solution, the absorbance of a colored solution, the rate of an enzyme-catalyzed reaction, or the radioactivity associated with a molecule.

If you measure a quantitative characteristic many times under identical conditions, a slightly different result will most likely be obtained each time.

If the absorbance of a solution is determined several times at a specific wavelength, the value of each measurement will surely vary from the others. Which measurements, if any, are correct?

Before this question can be answered, you must understand the source and treatment of numerical variations in experimental measurements. Analysis of Experimental Data. An error in an experimental measurement is defined as a deviation of an observed value from the true value. There are two types of errors, determinate and indeterminate, Determinate errors are those that can be controlled by the experimenter and are associated with malfunctioning equipment, improperly designed experiments, and variations in experimental conditions.

These are sometimes called human errors because they can be corrected or at least partially alleviated by careful design and performance of the experiment. Indeterminate errors are those that are random and cannot be controlled by the experimenter. Specific examples of indeterminate errors are variations in radioactive counting and small differences in the successive measurements of glucose in a serum sample.

Two statistical terms involving error analysis that are often used and misused are accuracy and precision. Precision refers to the extent of agreement among repeated measurements of an experimental value.

Accuracy is defined as the difference between the experimental value and the true value for the quantity. Since the true value is seldom known, accuracy is better defined as the difference between the experimental value and the accepted true value.

Several experimental measurements may be precise that is, in close agreement with each other without being accurate. If an infinite number of identical, quantitative measurements could be made on a biosystem, this series of numerical values would constitute a statistical population. The average of all of these numbers would be the true value of the measurement. It is obviously not possible to achieve this in practice. The alternative is to obtain a relatively small sample of data, which is a subset of the infinite population data.

The significance and precision of these data are then determined by statistical analysis. This section explores the mathematical basis for the statistical treatment of experimental data. Most measurements required for the completion of the experiments can be made in duplicate, triplicate, or even quadruplicate, but it would be impractical and probably a waste of time and materials to make numerous determinations of the same measurement.

Rather, when you perform an experimental measurement in the laboratory, you will collect a small sample of data from the population of infinite values for that measurement. To illustrate, imagine that an infinite number of experimental measurements of the pH of a buffer solution are made, and the results are written on slips of paper and placed in a container. It is not feasible to. By doing this, you have collected a sample of data. By proper statistical manipulation of this small sample, it is possible to determine whether it is representative of the total population and the amount of confidence you should have in these numbers.

The data analysis will be illustrated here primarily with the counting of radioactive materials, although it is not limited to such applications. Any replicate measurements made in the biochemistry laboratory can be analyzed by these methods. Radioactive decay with emission of particles is a random process. It is impossible to predict with certainty when a radioactive event will occur.

Therefore, a series of measurements made on a radioactive sample will result in a series of different count rates, but they will be centered around an average or mean value of counts per minute. Table 1. A similar table could be prepared for other biochemical measurements, including the rate of an enzyme-catalyzed reaction or the protein concentration of a solution as determined by the Bradford method. The arithmetic average or mean of the numbers is calculated by totaling all the experimental values observed for a sample the counting rates, the velocity of the reaction, or protein concentration and dividing the total by the number of times the measurement was made.

The mean is defined by Equation 1. The mean counting rate for the data in Table 1. If the same radioactive sample were again counted for a series of ten observations, that series of counts would most likely be different from those listed in the table, and a different mean would be obtained. If we were able to make an infinite number of counts on the radioactive sample, then a true mean could be calculated.

The true mean would be the actual amount of radioactivity in the sample. Although it would be desirable, it is not possible experimentally to measure the true mean. Therefore, it is necessary to use the average of the.

Since it is not usually practical to observe and record a measurement many times as in Table 1. This may be stated in the form of a question. How close is the result to the true value? One approach to this analysis is to calculate the sample deviation, which is defined as the difference between the value for an observation and the mean value, x Equation 1.

The sample deviations are also listed for each count in Table 1. Sample deviation. A more useful statistical term for error analysis is standard deviation, a measure of the spread of the observed values.

Standard deviation, s, for a sample of data consisting of n observations may be estimated by Equation 1. It is a useful indicator of the probable error of a measurement. Standard deviation is often transformed to standard deviation of the mean or standard error. This is defined by Equation 1. Equation 1. It should be clear from this equation that as the number of experimental observations becomes larger, sm becomes smaller, or the precision of a measurement is improved.

Standard deviation may also be illustrated in graphical form Figure 1. The shape of the curve in Figure 1. This mathematical treatment is based on the fact that a plot of relative frequency of a given event yields a dispersion of values centered about the mean, x.

The value of x is measured at the maximum height of the curve. The normal distribution curve shown in Figure 1. By using an equation derived by Gauss, it can be calculated that for a single set of sample data, Stated in other terms, there is a For many experiments, a single measurement is made so a mean value, X, is not known.

The parameter k is a proportional constant between Ex and the standard deviation. The percent proportional error may be defined within several probability ranges. Standard error refers to a confidence level of The constant k then becomes 1. The previous discussion of standard deviation and related statistical analysis placed emphasis on estimating the reliability or precision of experimentally observed values.

However, standard deviation does not give specific information about how close an experimental mean is to the true mean. Statistical analysis may be used to estimate, within a given probability, a range within which the true value might fall.

The range or confidence interval is defined by the experimental mean and the standard deviation. This simple statistical operation provides the means to determine quantitatively how close the experimentally determined mean is to the true mean. Confidence limits L and L2 are created for the sample mean as shown in Equations 1.

EquatiDn 1. The parameter t is calculated by integrating the distribution between percent confidence limits. Values of t are tabulated for various confidence limits. Each column in the table refers to a desired confidence level 0. The table also includes the term degrees of freedom, which is represented by n - 1, the number of experimental observations minus 1. The values of and sm are calculated as previously described in Equations 1.

The equations for statistical analysis that have been introduced in this chapter are of little value if you have no understanding of their practical use, meaning, and limitations. A set of experimental data will first be presented, and then several statistical parameters will be calculated using the equations. This example will serve as a summary of the statistical formulas and will also illustrate their application. Example 2 Ten identical protein samples were analyzed by the Bradford method for protein analysis.

The following values for protein concentration were obtained. Calculation of the sample deviation for each measurement gives an indication of the precision of the determinations, Standard deviation. The probability of a single measurement falling within these limits is For What personal protection items must be worn when handling glacial acetic acid? Draw a schematic picture of your biochemistry lab and mark locations of the following safety features: eyewashes, first-aid kit, shower, fire extinguisher, chemical spill kits, and direction to nearest exit.

Describe how you would prepare a l-liter aqueous solution of each of the following reagents: a 1 M glycine b 0. Describe how you would prepare just 10 mL of each of the solutions in Problem 4. If you mix 1 mL of the 1 M glycine solution in Problem 4 with 9 mL of water, what is the final concentration of this diluted solution in mM?

Convert each of the concentrations below to mM and f1M. You have just prepared a solution by weighing 20 g of sucrose, transferring it to a l-liter volumetric flask, and adding water to the line. These are standard concentration units used in the clinical chemistry lab. Convert the concentrations to mM. Further Reading Data Analysis P. Meir and R. Miller and]. Press Prospect Heights, IL. Reagents and Solutions M. Brush, The Scientist, June 8, , pp.

A discussion of water purification. Kegley and]. A discussion of water purity and analysis. Risley,] istry Lab. Schenectady, NY. Lowry and R. Lowry, Lowry Chem. Safety in Academic Chemical Laboratories, 6th ed. Writing Laboratory Reports K.

Covers several important ing lab orientation, keeping a notebook, and lab procedures. Cummings Phoenix. Menlo Park, CA. Beall and].