Solution Manual For Experiments in Biochemistry: A Hands-on Approach, 2nd Edition

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iManual forExperiments inBiochemistry:A Hands On ApproachShawn O. FarrellandLynn E. TaylorColorado State University

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iiTable of ContentsPreface ....................................................................................................................... iiiTechnical Support...................................................................................................... ivBackground Information from Textbooks................................................................. vGeneral Lab Equipment............................................................................................. viExperiment 1 -- Use of pipettors ............................................................................... 1Experiment 2 -- Preparation of Buffers ..................................................................... 7Experiment 3 -- Beer's Law and Standard Curves .................................................... 14Experiment 3a -- Protein Concentration of LDH Fractions ...................................... 20Experiment 4 -- Purification of LDH (short version)................................................ 25Experiment 4a -- Purification of LDH (comprehensive version).............................. 33Experiment 5 -- Separation and Identification of Amino Acids ............................... 42Experiment 5a -- Purification of LDH with Ion Exchange Chromatography ........... 49Experiment 6 -- Affinity Chromatography of LDH .................................................. 57Experiment 6a – Affinity Chromatography of LDH ................................................. 61Experiment 7 -- Gel Filtration Chromatography....................................................... 69Experiment 7a -- Gel Filtration Chromatography of LDH........................................ 74Experiment 8 -- Enzyme Kinetics of Tyrosinase ...................................................... 82Experiment 8a -- Enzyme Kinetics of LDH .............................................................. 89Experiment 9 -- Native Gel Separation of LDH Isozymes (short version) ............... 96Experiment 9a -- Native Gel Separation of LDH Isozymes (comprehensive).......... 101Experiment 9b -- SDS-Polyacrylamide Gel Electrophoresis (short)......................... 106Experiment 9c -- SDS-Polyacrylamide Gel Electrophoresis (comprehensive)......... 112Experiment 10 -- Western Blots of Serum Proteins .................................................. 119Experiment 10a -- Western Blot of LDH .................................................................. 127Experiment 11 -- Analysis of DNA Restriction Fragments ...................................... 134Experiment 12 -- Cloning and Expression of Barracuda LDH ................................. 141Experiment 13 -- Polymerase Chain Reaction .......................................................... 158

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iiiPrefaceThis manual is designed to aid the laboratory instructor in preparing for, supervising, andanalyzing experiments inExperimental Biochemistry: A Hands On Approach.The textwas written with the idea of its application to a wide variety of biochemistry lab courses.Whether your course is for non-majors and meets only once a week for 3 hours, or is amore intensive course requiring several meetings, you should be able to find theappropriate length and intensity of experiments. Many of the experiments are suitable fora survey course and can stand alone. For those interested in a comprehensive approach,there are groups of experiments that do make an integrated package. Many of thetechniques used offer both types of experiment. For example, chapter 7 involves gelfiltration chromatography. Experiment 7 is a stand alone experiment involving estimatingthe molecular weight of myoglobin with SephadexG-75. Experiment 7a is part of thecomprehensive purification of lactate dehydrogenase and uses gel filtration on SephadexG-150 or Sephacryl S-200 as the final purification step.Each chapter of thismanualcorrespondsto thesamechapterinExperimentsinBiochemistryand includes the following:Estimated time required by competent and prepared studentsMaterials needed for the experiment and sources when necessaryHints for the preparation of reagents and equipment, and pitfalls to avoidSuggestions for disposal and clean up of experimentsSample data the students are likely to see and answers to the end of experimentcalculationsAnswers to the additional problem sets.Wehopethatarmedwiththismanualyouwillfindtheimplementationoftheexperiments straight forward. If you ever have trouble, please feel free to contact theauthor (see Tech Support).

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ivTechnical SupportI have been teaching two levels of biochemistry lab at Colorado State University for thepast 18 years. One is a survey course for non-majors and the other is a comprehensivecourse for majors. My position is pure teaching, so I have devoted much time to theperfection of these experiments.If you have any questions about how to implement any of these experiments, how toprepare the reagents, where to get the reagents and equipment, how to optimize the timeyou have with your course, or what might have gone wrong, please feel free to contactme. I am available by phone or e-mail. I would also appreciate any feedback you give methat would make the labs run smoother. Thank-you and I look forward to hearing fromyou.Dr. Shawn O. FarrellUSA Cycling1 Olympic PlazaColorado Springs, CO 80909719-866-3364sfarrell@usacycling.org

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vBackground Information from TextbooksNo biochemistry laboratory can stand alone without a supporting lecture class to give thestudents the background material. We have attempted to include material in our chaptersthat is specific to laboratory techniques. For background material of a broader nature, wesuggest one of the following textbooks --Biochemistryby Mary K. Campbell and ShawnO. Farrell orBiochemistryby R.H. Garrett and C.M. Grisham. Of course, any basicbiochemistry book would also do.Below you will find information about the relevant chapters of the fifth edition ofCampbell and Farrell'sBiochemistryand the third edition of Garrett and Grisham'sBiochemistry.Experiments in BiochemistryCampbell (5th edition)Garrett and Grisham (3rd edition)Chapter 2Chapter 2Chapter 2Chapter 3Chapter 5Chapter 4Chapters 4,5, 6Chapters 5,6,13Chapter 5Chapter 3Chapters 4,5Chapter 6Chapter 5Chapter 5Chapter 7Chapter 5Chapter 5Chapter 8Chapter 6,7Chapters 13,14Chapter 9Chapter 5,13Chapter 5Chapter 10Chapter 12Chapter 11Chapter 13Chapter 12Chapter 12Chapter 13Chapter 12Chapter 13Chapter 13Chapter 12

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viGeneral Lab EquipmentThe following chapters contain lists of equipment required for each experiment. Thereare certain items that are assumed to be available for all labs, and these will not bementioned specifically in the chapters. These items include the following:KimwipesglovesPasteur pipets (long and short)pipet tips (all sizes)Parafilmpaper towelssoapvortexersweigh boatsice bucketsmicrocentrifuge tubesThe quantities listed are for each experiment performed. Thus, if you have students workalone, that would be per student. These are the quantities that I have used. Sometimes theyare overkill, but I prefer to err on the side of making too much of the cheaper chemicals sothat I don't have to make them again later.

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1Chapter 1 -- Biochemistry Boot CampExperiment 1 -- Use of PipettorsTime RequiredThis brief experiment should take no more than 30 minutes, usually less if there is ampleequipment.Materials RequiredP-100 or P-200 pipetmen(Rainin, Integrapette, Eppendorf)P-1000 pipetmenTop loading balances or others with a sensitivity of at least 0.01gDeionized waterHints for Preparation and ImplementationIf equipment is limiting, students can stagger which part they start first to better sharethe pipetmen.Be sure to give a prelab lecture about how to use a pipettor. Many students, includinggrad students, have never received any formal training in this. This lab, though brief,will establish the quality of results that will be seen for the rest of the semester.Make sure that students identify poorly calibrated pipets.If a student thinks that their pipettor draws up a low volume, make sure the pipet tipsare on tight, the pipet is set to the correct volume, and that the lower barrel of thepipettor is screwed on tight. The latter is a common problem with Eppendorfpipettors.Waste DisposalNoneAnswers to Prelab Questions1.What is the useable range of a P-1000 Pipetman?100 to 1000L

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22.What is the difference between accuracy and precision?Accuracy is the relationship between the number you read and the true value. Inother words, if you try to pipet 1 mL and you actually pipet 0.99 mL, then thepipetting was accurate. Its error was only 1%. Precision is how reproducible thenumbers are, so if you try to pipet 1 mL five times and you pipet 0.70, 0.70, 0.70,0.70, and 0.70, then your pipetting was very precise although it was also veryinaccurate.3.What should 100L of water weigh?Since water weighs 1 g/mL, the weight would be calculated thusly:1 g/mL x 0.1 mL =0.1 g4.What should 1000L of water weigh?The answer is 1 g, since 1000L is the same as 1 mL.Sample Data and Analysis of ResultsPart A -- Precision of P-100 or P-200 pipettors1.Record the weight you measured for the three trials of 100L:Weight #1 (x1) 0.09Weight #2 (x2) 0.09Weight #3 (x3) 0.112.Average the three weights.Average of three trials:0.0973.Calculate the % error between the average of the three trials and the truevalue.% error =│0.097 - 0.100 g│x100 = 3 %0.1 g4.Calculate the mean deviation for the three trials:mean deviation =│xi- xavg│=3[-(0.09-0.097)+-(0.09-0.097)+(0.11-0.097)]/3 =0.009

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3Part B -- Precision of P-1000 pipettors1.Record the weight you measured for the three trials of 1000L:Weight #1 (x1) 0.99Weight #2 (x2) 1.05Weight #3 (x3) 0.972.Average the three weights.Average of three trials:1.003.Calculate the % error between the average of the three trials and the truevalue:% error =│avg. weight - 1.00 g│x100 =01.00 g4.Calculate the mean deviation for the three trials:mean deviation =│xi- xavg│=3[-(0.99-1.00)+(1.05-1.00)+-(0.97-1.00)]/3=0.035.Record the weight you measured for the three trials of 100L using the P-1000:Weight #1 (x1) 0.09Weight #2 (x2) 0.08Weight #3 (x3) 0.126.Average the three weights.Average of three trials:0.107.Calculate the % error between the average of the three trials and the truevalue:% error =│avg. weight - 0.10 g│x100 =00.10 g

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48.Calculate the mean deviation for the three trials:mean deviation =│xi- xavg│=3[-(0.09-0.10)+-(0.08-1.00)+ (0.12-0.10)]/3=0.02Part C – Pipettors in the Lab1.Which of the two pipettors you used was the more accurate?The % error is the measure of the accuracy of a pipetman. From the data presented,the P-1000 would appear to be the more accurate, since it had the smallest % error.2.Which of the two pipettors you used was the more precise?This is the trickier question. If you just look at the mean deviations, it appears thatthe P-1000 has the larger mean deviation of 0.02 when compared to the 0.009 of theP- 100, when both were used to pipet 100L. However, one must bear in mind thatthe total weight expected is also important. For the pipetting of 1000L, if youcalculated a % mean deviation by dividing by the expected weight and multiplyingby 100, the P-1000 would, once again, have the smaller number.3.What are the take-home messages from this exercise?Take your pick from any of the following:There are different types of pipetmen that you must learn to use.Pipetmen are precise in the hands of a trained user, but not necessarily accurate.The accuracy of a pipet should be checked frequently.It is easy to check the accuracy and precision by doing a water weight test.Pipetmen have a range outside of which they are not accurate.You should learn what the correct volume of solution looks like in a pipet tip.Others that you can think of.4.Without checking the accuracy of a given Pipetman, would you predict that it isbetter to use a P-200 or P-1000 to pipet 100 μL? Why?It is generally better to use a liquid transfer device closer to its maximum volume, sousing the P-200 would be better.

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55.Is a Pipetman more like a serological pipet or a Mohr pipet? Why?It is more like a serological pipet, as you expel the liquid completely out of it all theway to the tip.6.If you are trying to pipet an unknown liquid with a Pipetman and the liquidkeeps running out of the tip before you can transfer it, what are two possiblereasons for this? What can you do to remedy the situation?One reason might be that the tip is not on tightly. In that case, just tighten the tip.Another might be that the liquid is an organic solvent, which might have a very lowsurface tension. To remedy that, draw up the liquid into the tip and then expel it.Then draw up the solution again. It usually will hold in the tip the second time afterpre-wetting the tip.7.How do you make 200 mL of a 0.1 M solution of a substance that has amolecular weight of 121.1 g/mol?You need 0.2 liters of a 0.1 mole/liter solution or 0.02 moles of the solute. If theMW is 121.1 g/mol, you need 0.02 x 121.1 or 2.4 grams of solute. Thus, you weigh2.4 grams of solute into a vessel and bring the volume up to 200 mL.8.If you take 10 mL of the solution you made in Question 7, add 90 mL of water,mix, and then take 5 mL of the mixture and bring it to 25 mL, what will be theconcentration of the final solution in molar, millimolar, and micromolar?The first dilution is a 10 to 1, since you start with 10 mL and end with 100 mL. Thesecond dilution is a 5 to 1, since you took 5 mL and brought the volume to 25. Thus,the total dilution factor is 50 to 1. Since you started with 0.l M, the finalconcentration in molar is 0.1/50 or 0.002 M. this is 2 mM and 2000 μM.Answers to Additional Problem Set1.How many grams of solid NaOH are required to prepare 200 mL of a 0.05 Msolution?0.4 g2.What would be the concentration from Problem 1 expressed in % w/v?0.2 % w/v3.How many mL of5M NaCl are required to prepare 1500 mL of 0.002 MNaCl?0.6 mL

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64.What would be the concentration of the diluted solution from Problem 3expressed in mM,M, and nM?2 mM, 2000M, 2 x 106nM5.A solution contains 15 g of CaCl2in a total volume of 190 mL. Express theconcentration in terms of g/L, % w/v, M, and mM.79 g/L, 7.9 % w/v, 0.71 M, 710 mM6.Given stock solutions of glucose (1M), Asparagine (100 mM) and NaH2PO4(50mM), how much of each solution would you need to prepare 500 mL of areagent whichcontains0.05 M glucose, 10mM Asparagineand 2mMNaH2PO4?25 mL glucose, 50 mL Asparagine, 20 mL sodium phosphate7.Calculate the number of millimoles in 500 mg of each of the following aminoacids: alanine (MW = 89), leucine (131), tryptophan (204), cysteine (121), andglutamic acid (147).5.6 mmol Ala, 3.8 mmol Leu, 2.5 mmol Trp, 4.1 mmol Cys, 3.4 mmol Glu8.What molarity of HCl is needed so that 5 mL diluted to 300 mL will yield 0.2M?12 M9.How much 0.2 M HCl can be made from 5.0 mL of 12.0 M HCl solution?300 mL10.What weight of glucose is required to prepare 2 L of a 5% w/v solution?100 g11.How many mL of an 8.56% solution can be prepared from 42.8 g of sucrose?500 mL12.How many mL of CHCl3are needed to prepare a 2.5% v/v solution in 500 mLof methanol?12.5mL13.If a 250 mL solution of ethanol in water is prepared with 4 mL of absoluteethanol, what is the concentration of ethanol in % v/v?1.6 % v/v

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7Chapter 2 – Acids, Bases, and BuffersExperiment 2 – Preparation of BuffersTime Required1.5 to 2 hours if there is no equipment constraint.Materials RequiredpH meters, preferably digitalNaOH, 1 M15 mLHCl, 1 M15 mLNaAcetate1 gCAPS“HEPES“Na2HPO4“Tricine“TRIS“NaCitrate“NaH2PO4unknowns,0.1 M, pH 5.65 mlNaH2PO4unknowns,0.1 M, pH 7“NaH2PO4unknowns,0.1 M, pH 8“Sources for key ingredientsNone of the chemicals used for this experiment need to be particularly pure. Use thecheapest source you can find. For the buffer compounds, it does not matter if you use theacid form or the basic form, as the students will adjust the pH anyway when making up thebuffer. Sigma and VWR offer good sources of these compounds. For convenience, theSigma materials are listed below:CompoundCatalog #Price (2004)Acetate (sodium salt)Sigma 24,124-5$64.30 per KgCAPSSigma C 2632$243 per KgCitrate (sodium salt)Sigma C 3674$27.60 per KgHEPES (sodium salt)Sigma H 7006$216.50 per 500 gTricineSigma T 0377$86 per 250 gTRIS (Trizma base)Sigma T 1503$84 per Kg

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8Hints for Preparation and ImplementationThis is a fast lab that usually only slows down due to the logistics of acquiring the materialsand using the balances and pH meters.Make sure all available pH meters and balances are ready to go.Dispense the dry buffer powders so that there are several locations or several containersstudents can use to avoid a big line of students waiting to get started.Dispense unknowns in large test tubes with about 10 mL each.Waste DisposalAll unknowns can be dumped down the sink at the week’s end. Save the HCl and NaOHfor future labs. Discard or save dry buffers depending on quality and quantity.Answers to Prelab Questions1.Calculate the weight of buffer you would use to make the buffer for part A forthe six possibilities:0.1 mol/liter x 0.1 liters = 0.01 moles. You need 0.01 moles of the buffer. Moles xMW = grams.Acetate136.1 g/mol x 0.01 mol = 1.36 gCAPS221.3 g/mol x 0.01 mol = 2.21 gCitrate294.1 g/mol x 0.01 mol = 2.94 gHEPES238.3 g/mol x 0.01 mol = 2.38 gPhosphate142.0 g/mol x 0.01 mol = 1.42 gTricine179.2 g/mol x 0.01 mol = 1.79 gTRIS121.1 g/mol x 0.01 mol = 1.21 g2.If we give you HEPES in the basic form and ask you to make a buffer of pH8.0, will you add HCl or NaOH to adjust the pH? Why?Calculate what would be the initial pH of a solution of HEPES base. Using theformula:pH = (pKa + 14 + log[base])/2=(7.55 + 14 + log[0.1])/2 = 10.28We can see that the pH will be more basic than the desired 8.0. Therefore, we willhave to add acid. In general, anytime you make up a buffer in its basic form, youwill need to add acid to get to a useable buffer pH. This question could be answeredphilosophically as well. A buffer always needs to be a combination of the acid andbasic form of the buffer. If we start with just the basic form of a buffer, we willalways need to create the acidic form, so we would need to add HCl.

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9Sample Data and Analysis of ResultsDataPart ABuffer #1:phosphategrams: 1.42Original pH:5.4Buffer #2:Tricinegrams: 1.79Original pH:10.2Part BBuffer #1pH of 0.1 M8.10pH of 0.01 M 8.40pH of 0.001 M8.75Buffer #2pH of 0.1 M8.10pH of 0.01 M7.99pH of 0.001 M7.95Part CDistilled water pH:5-9 (depending on water source and cleanliness of beakersUnknown #21pH:7.0Unknown #36pH:5.6Part DBuffer chosen:TricinepH:8.10pKa:8.15Acid or base added:NaOHpH after adding acid or base:8.18pH of 50 mL of water: 7.0pH after adding acid or base:12.0Calculations1.(a)What is the ratio of A-/HA in your buffer after you adjusted its pH to therequired value?This will depend on which buffer you used and at what pH. A good buffer wasdefined as one with a pH that was at most 1 pH unit away from the pKa.

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