Biology 309 Lab Notebook

Name: Biology 309 Lab Notebook This is a guided lab notebook for you to keep well-organized notes about procedures and record experimental data for experiments as they are performed. It is guided because, instead of providing a notebook with blank pages, you are given a framework for recording certain key information. What you are required to include is not the thorough record that would be expected in an actual research laboratory, but will model how such notes should be maintained. Note keeping guidelines Notes must be neat. If you can do so in ink, fine. But if not, then you must use a pencil. When necessary continue not taking on back side of pages. The first page of each section must be completed before coming to lab, and will be collected at the start of lab. If you are working on it in lab it will not be accepted. Completing this page is critical to your preparation for the lab exercise. Other pages of the lab notebook will be collected periodically. Recording Procedures: Each student is expected to take notes as procedures are performed. Recording Data: In some labs, it will be easier for one student to record the data as the partner carries out a procedure. This is fine, but the data should be copied over to the other lab notebook. All graphs should be fully and properly formatted per the Figure preparation guidelines. Graphing, calculations, and other data processing: must be done but each student individually; although you are expected to compare notes to assure agreement after doing so. Other than to transfer data, copying from another person s notebook will be considered plagiarism. Solution Preparation It is expected that both group members participate in the preparation of all solutions. Table of contents Solution Preparation -- page 3 Histology -- page 9 PET and Hill reaction -- page 13 Culturing B16 cells -- page 23 Protein measurement -- page 31 Gel Electrophoresis -- page 39 Immunoblotting -- page 45 Page 1

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Name: Solution Calculation and Preparation 1. What is the equation that is used when diluting a reagent solution to a lower concentration? 2. What is the equation that is used for calculating the grams of a dry reagent for a molar concentration solution? 3. What is the equation used when calculating the grams of a dry reagent for a %(w/v) solution? 4. What is the equation used when calculating the volume of a liquid reagent for a %(v/v) solution? For Hill Reaction lab Homogenization buffer (50 ml) Prepare before lab day Calculations for Tricine: for Sucrose for NaCl: Components Final conc. MW / 50 ml Tricine-NaOH 1 50 mm g Sucrose 0.4 M g NaCl 10 mm g ph 7.8 1 The designation "-NaOH" after Tricine indicates that the ph of the solution should be adjusted with NaOH. Prepare by sequentially dissolving Tricine, Sucrose and NaCl in 40 ml of uph 2 O in a 100 ml beaker on a magnetic stirrer. Adjust the ph with NaOH (first with 10M, and then with 1M). Transfer to a 50ml graduated cylinder and bring volume up to 50 ml with uph 2 O. Store solution in the refrigerator. Page 3

For Histology lab 1. Isopropyl alcohol (IPA) solutions solution volume of IPA Volume of H 2 0 250 ml of 70% (v/v) IPA ml ml 50 ml of 85% (v/v) IPA ml ml 200 ml of 95% (v/v) IPA ml ml For each solution, add the IPA to a graduated cylinder, bring volume up to the indicated final volume with roh 2 O, and transfer to a reagent bottle. Calculations for 70%: for 85%: for 95%: 2. Scott s solution (100 ml) Final Components conc. MW / 100 ml NaHCO 3 240 mm MgSO 4 1.7 mm Calculations For NaHCO 3 : For MgSO 4 : Page 4

Name: For Protein Measurement/Spectrophotometry lab 1. Homogenization buffer (100 ml) Prepare before lab day Reagents Final Conc. MW / 100 ml Tris-HCl 1 50 mm g 20 mm EDTA 2Na 1 mm NA ml ph 7.5 1 In powdered form Tris is sometimes labeled as "Trizma or Trizma-base". The designation "- HCl" after Tris indicates that the ph of the solution should be adjusted with HCl. Calculations for Tris: for EDTA: Prepare by dissolving Tris in 80 ml of uph 2 O in a 250 ml beaker on a magnetic stirrer. Then pipet in the appropriate volume of EDTA Adjust the ph with HCl (first with 10M, and then with 1M). Transfer to a 100ml graduated cylinder and bring volume up to 100 ml with uph 2 O. Store in a 125 ml reagent bottle in the refrigerator. 2. BCA reagent (30 ml) (you will not actually prepare this solution) Components Final conc. / 30 ml BCA reagent A 100% 30 ml 4% CuSO 4 (w/v) 0.08% ml Calculations for CuSO 4 : The BCA reagent is prepared by mixing the appropriate amount of 4% CuSO 4 with Reagent A, which contains a proprietary mixture of bicinchoninic acid and other chemicals. Page 5

For Electrophoresis and Electroblotting lab (2 solutions) 1. Reservoir Buffer (500 ml) Prepare before lab day Components Final Conc. MW / 500 ml Tris 1 25 mm g Glycine 200 mm g SDS 2 0.1% (w/v) NA g 1 In powdered form, Tris is labeled as "Trizma or Trizma-base". 2 SDS is an abbreviation for sodium dodecyl sulfate (or sometime sodium laurel sulfate ). Calculations for Tris: for Glycine: for SDS: Prepare by sequentially dissolving Tris, Glycine and SDS in 400 ml of uph 2 O in a 1 L beaker on a magnetic stirrer. The ph of this solution should not be adjusted, since the quantities of Tris and glycine added result in an appropriate ph (8.3). You should check the ph and see that it falls reasonably closed (±0.5 ph unit) to this value. Transfer to a 500 ml graduated cylinder, bring volume up to 500 ml with uph 2 O, and store in a 500 ml reagent bottle. 2. Transfer buffer (Liter) Prepare before lab day Components Final conc. MW / L Tris 1 25 mm g Glycine 200 mm g Methanol 20 % (v/v) NA ml 1 In powdered form, Tris is labeled as "Trizma or Trizma-base". Calculations for Tris: for Glycine: for Methanol: Prepare by dissolving the Tris and glycine in 600 ml of uph 2 O in a 1L beaker on a magnetic stirrer. Add the appropriate volume of methanol, and then bring solution up to its final volume. As for the above solution, do not adjust the ph. Store this solution a 1L reagent bottle in the refrigerator. Page 6

Name: 3. Sample Denaturation Buffer (you will not actually prepare this solution) Components Final Conc. MW / 25 ml Stacking gel buffer 40% v/v NA ml glycerol 10% w/v NA g SDS 2% w/v NA g 0.1% w/v BPB 1 0.01% w/v NA µl βme 2 6% v/v NA ml 1 = Brome-phenol-blue 2 = β-mercaptoethanol Calculations for Stacking gel buffer: for Glycerol: for SDS: for BPB: for Βme 2 : For Immunoblotting Lab 1. Wash buffer (150 ml) - prepare before lab day Components Final conc. MW / 150 ml Tris-HCl ph 7.5 10 mm g NaCl (powder) 150 mm g 1% Tween-20 1 0.05% v/v NA ml 1 Tween-20 is a mild detergent that reduces non-specific binding of antibodies to the nitrocellulose membrane and contributes to membrane blocking. Calculations for Tris: for NaCl for Tween-20: Prepare by dissolving Tris and NaCl in 120 ml of uph 2 O in a 250 ml beaker on a magnetic stirrer. Then pipet in the appropriate volume of Tween-20. Adjust the ph with 1 M HCl. Transfer to a 250ml graduated cylinder and bring volume up to 150 ml with uph 2 O. Store in a 250 ml reagent bottle. Page 7

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Name: Histology exercise What is the purpose of this lab exercise? Tissue preparation Describe the tissues ressected from the mouse and how they were handled Actual fixation, dehydration and infiltration schedule Solution Date & time-in Date and time-out Time length (hr) Identify each of the tissue blocks that you prepare Block # Tissue Orientation Page 9

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Name: Histology exercise, cont. You are expected to track precisely the ribbons that you cut; tissue, thickness, orientation Slide preparation Staining time Slide # Tissue Thickness Hemotox. Eosin Describe the qualities of the two best slides (one for each tissue) you prepare -- condition of tissue; distinct tissue structures you were able to identify Slide # and tissue: Page 11

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Name: Studying PET through the Hill Reaction Answer questions A - D before coming to lab A. What is the purpose of this exercise? B. Why are chloroplasts used in this exercise instead of purified thylakoids? C. What is the hypothesis stated in the lab exercise about the location where DCPIP absorbs electrons? D. Based upon this hypothesis, which of the inhibitors should block DCPIP reduction in the Hill reaction? Explain your reasoning for each of the three inhibitors. E. Why is it necessary to include measurements of chlorophyll concentration and light intensity in the calculation of the Hill reaction? Page 13

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Name: Studying PET through the Hill Reaction Recording of protocols and data For these protocols Chloroplast preparation Chlorophyll measurement Light measurement Record each step of the procedures, focusing on specific volumes, quantities, or other quantitative parameters, and stating the purpose for that step. Be sure to record critical values such as the chlorophyll absorbance measurement and light intensity measurement. Hill reaction assays: Record general steps of the protocol, focusing on specific volumes, quantities, or other quantitative parameters, and stating the purpose for that step. For all assays, record the absorbance measurements made in tables as illustrated on the next page. Indicate how protocol was varied for control and inhibitor assays. Page 15

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Name: Studying PET through the Hill Reaction Recording of Hill reaction data Create tables like this to record your raw data Assay Description: Modifications, etc.: Min Abs 0 0.5 1 1.5 etc Page 17

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Name: Studying PET through the Hill Reaction Results calculations Show calculations clearly and neatly is spaces provided A. Using the Beer-Lambert law (abs = concentration x abs coef.), calculate the chlorophyll concentration of your chloroplast suspension. The absorption coefficient for chlorophyll at 652 nm is 35.4 ml cm-1 mg-1. ABS 652 = dilution factor = [chlorophyll] (and units) = (value should be between 0 and 10.) B. Following the instructions in the manual and using the above value, calculate the mg of chlorophyll in the chloroplast suspension added to each Hill assay mixture: mg chlorophyll in assay C. Following the instructions in the manual, and the light intensity measured in lux, calculate the photon fluence rate: Measured light intensity: lux Photon fluence rate (and units) = D. Determine the rate of DCPIP reduction Slope 1 (and units) = Page 19

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Name: Studying PET through the Hill Reaction Results calculations, cont. E. Following the instructions in the manual, complete the calculation of the Hill reaction rate. Show calculations for each assay: Rate 1: (correct calculations should yield a number between 0 and 1) Average rate (and units): Control: What was effect of eliminating light? Inhibitors What was the effect of adding each inhibitor? Page 21

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Name: Culturing and Fluorescent Staining of B16 melanoma cells Week 1 - Culturing and Passaging of cells Answer questions A - E before coming to lab A. What is the purpose of this lab exercise? B. Is your B16 cell culture a primary or secondary culture? Explain. C. Why is it necessary to passage cultured cells periodically; explain all the reasons why this is necessary. D. What are the 3 components of CDMEM and what are their functions? E. Why is Trypsin/EDTA used during the passaging process? Page 23

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Name: Culturing and Fluorescent Staining of B16 melanoma cells Passaging record Date of passage: % Confluence: Name of person(s) doing passaging: ml of Trypsin/EDTA used: Incubation time at 37 O C: Cell Counts: #1 #2 Calculation of cell density: Calculation of volume containing 10 4 cells: Totals: Avg: Replicate this table below for each passaging Page 25

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Name: Culturing and Fluorescent Staining of B16 melanoma cells Week 3 - Rhodamine-phalloidin staining Answer questions A - D before coming to lab A. What are f-actin and g-actin? How are they related? B. What is the purpose for each of these reagents in the staining process? Rhodamine: Phalloidin: Fixative: Permealization buffer: DAPI: C. What is the fundamental property of a molecule that acts as a fluorochrome? D. In the fluorescence microscope, what are the functions of the Barrier filter: Excitation filter: Page 27

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Name: Culturing and Fluorescent Staining of B16 melanoma cells Rhodamine-phalloidin staining, cont. Record the steps of the procedure as you perform them; the emphasis should be on the quantities, time periods, volumes Page 29

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Name: Protein Measurement through Absorption Spectrophotometry Answer questions A - D before coming to lab A. Why are you preparing a liver homogenate in this lab exercise; how will it be used later in the upcoming lab exercises? B. What causes light scattering and how does it affect absorbance measurements? C. Explain how a protein standard curve is created and used to determine the protein concentration of an unknown. Include in you explanation, the purposes of BCA and BSA. D. In protocol #2, Measuring the protein concentration of the homogenate, you will dilute your liver homogenate. Why? Determine the volumes that you will need to use and right them here and in the lab manual is the space provided. Page 31

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Name: Protein Measurement through Absorption Spectrophotometry Procedures You can cite the procedures from the lab manual, ( Procedure as described in. ) but record all the quantitative values (weight of liver, length/speed of centrifugation, volumes of solutions) and record any deviations from the cited procedure. be thorough! Page 33

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Name: Protein Measurement through Absorption Spectrophotometry Results Print on or tape to the back of pages your final graphs Complete the following tables to record your results Table 1. Data for BSA protein assay Tube # μg/μl BSA Abs @ 562 nm 1 Slope of trendline = :L cm -1 :g -1 Table2. Data for Liver homogenate replicas μg/μl protein Replica # Abs @ 562 nm (from standard curve) (blank) Tara to 0 0 1 Avg ------ μg/μl Protein concentration in the original homogenate = Volume of homogenate containing 50 μg protein: μl **Copy this value to page 4 (A.Sample preparation, step 3B) of the Electrophoresis and Electrooblotting lab exercise** Page 35

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Name: Protein Measurement through Absorption Spectrophotometry Results, cont. Table 3. Absorption spectrum measurements (20 nm increments) Absorbance Absorbance Wavelength BCA only BCA + BSA 420 nm 8 max lies between and nm Table 4. Absorption spectrum measurements (5nM increments) Absorbance Absorbance Wavelength BCA only BCA + BSA Observed 8 max for BCA reagent + protein: Page 37

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Name: Gel electrophoresis and electroblotting Answer questions A - F before coming to lab A. Purpose of this and next week s exercises: B. Identify the three protocols in this and next weeks labs and their purposes: 1. 2. 3. C. What is the concentration of protein in your liver homogenate: D. What is the volume of homogenate containing 50ug of protein (show calculations): E. Give the volumes of each component that will be combined to prepare your liver homogenate for electrophoresis: A. μl SDB B. μl liver homogenate C. μl VAB F. What are the functions of these components of the SDB: SDS: Brome-phenol-blue: Β-mercaptoethanol: Page 39

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Name: Gel electrophoresis and electroblotting, cont. Create a table with each lane number, the samples that were loaded and how they will be later treated: (although these specific lane number should not be included in the lab report). Lane sample eventual treatment How long was the gel run for, at what current: For how long was electroblotting performed, and at what current Describe the appearance of your Amido black stained gel Create here the table for data from the MW Standard Curve (see Table 1) Page 41

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Name: Review questions: Type responses 1. Why do the peptides move through the gel only according to their molecular weight rather than due to charge, shape and subunit structure? 2. What factors (other than the properties of the proteins themselves) would effect the rate of movement of the proteins through the gel. 3. What is a dalton and approximating how many AAs are present in a protein of 50 Kd? Show how this was calculated. 4. During the electroblotting procedure what would be the result if the orientation of the polyacrylamide gel and the nitrocellulose membrane were inadvertently reversed relative to the electrodes? 5. Why must the proteins be electroblotted to nitrocellulose before probing with antibodies; i.e., why do we not probe the gel directly? 6. Why is it necessary to block the nitrocellulose membrane before performing the Western blotting procedure? What would happen during the immunoblotting procedure if this step were left out? Page 43

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Name: Immunoblotting Answer A D before coming to lab A. Blocking buffer: purpose: B. Primary Ab: purpose, type, concentration, and incubation time: C. Secondary Ab conjugate: purpose, type, concentration, and incubation time: D. Staining reagent: type, explain why it changes color: Page 45

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Name: Immunoblotting, cont. Identify the treatment used for your membrane: Describe the appearance of your membrane (or if yours was a negative control, than that of another group with bands): In the space below identify each control run in this lab exercise, the alternative explanation it is designed, and which bands appeared 1. 2. etc Page 47

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Name: Immunoblotting, cont. Copy Table 1 for immunoblotting results below. What are the specific conclusions drawn about the results during the class discussion Page 49

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Name: Review questions: Type responses 1. How is a primary antibody, such as rabbit anti-human β-actin prepared? 2. What is goat-anti-rabbit IgG alkaline phosphatase? 3. What is the advantage of using secondary antibodies, rather than simply attaching a detection enzyme such as alkaline phosphatase directly to the primary antibody? 4. What was the conclusion drawn for the experiment relevant to the hypothesis. Write a numbered list of the evidence that supported this interpretation. 5. Some of the bands in Western blots were not actin. What caused these bands to appear? 6. We identified one of the aberrant proteins; what was it? Write a numbered list of the evidence that supported this interpretation. Page 51

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