In March 2001, a Gallup poll asked, “How would you rate the overall quality of the environment in this country today—as excellent, good, fair or poor?” Of 1060 adults nationwide, 46% gave a rating of excellent or good. Is this convincing evidence that a majority of the nation’s adults think the quality of the environment is fair or poor? Test using α = .05.
Study Guide Lab Exam 1 LAB 1: Digital Microscopy of Live Cells 1. Compare and contrast brightfield, darkfield, and phase-contrast optics. Brightfield: background is brightly lit and only colored specimens that absorb light at particular wavelengths/reflect or refract other wavelengths produce any contrast; best used to view dead stained cells or live cells with naturally-occurring pigments (though most live cells are nearly transparent and lack sufficient contrast to be adequately seen ((with brightfield optics.)) Condenser should be lowered as much as possible. Darkfield: background is dark/black and only light from the outer edge of the light source (from a disc placed in the condenser) that surrounds the specimen, making it bright white, is allowed to reach the specimen (instead of light coming up through the specimen.) Phase-Contrast: background is dimly-lit (dark grey) that allows light refracted by structures in a transparent specimen to stand-out as white (in phase) or black (out of phase) (by a special condenser coupled with special objectives); for the first time allowed the study of internal cell structure without the need to stain/kill the cells. Condenser should be higher, but not the highest. 2. Explain how quantitative data were collected from images. By use of a microscope equipped with a digital video camera interfaced to the computer; use the camera to capture still and video images (Motic Images Plus 2.0ML) and software (ImageJ) to analyze the images. The colored images are turned to 8-bit grayscale images, the line tool is dragged horizontally across the cells, and a Plot Profile graph is made showing the gray level values that range from 0(black)-255(white) (with greys in between 0- 255.) These values are then entered on Excel in the form of XY scatter plots with the Y-axis representing the gray values and the X-axis representing the pixel numbers. 3. Draw and label representative graphs of line plots of gray values from images of cells taken with different optics. BF more white PC varying values DFblack, whites/greys, black 4. Explain how line plots of gray values show contrast in images. The more variation in the gray value range there are, the more contrast the image shows. 5. Identify the parts of the light microscope and give a function of each. Eyepiece & Binocular Eyepiece tube: the lens you look through to see your specimen & adjuster of focus on eyepiece. Interpupillary distance scale: adjusts how far apart lenses are for eyes. Objectives (4X, 10X, 40X): most microscopes have 2, 3, or more lenses that magnify at different powers. Always start with the lowest power and work your way up to the strongest when examining a specimen. The shortest lens is usually the lowest power. Specimen Holder & Stage: holds the specimen in place & is where the sample/specimen is placed for viewing. Aperture: It’s the hole in the stage that allows light through for better viewing of the specimen. Condenser: a lens that serves to concentrate light from the illumination source that is focused through the object and magnified by the objective lens. Coarse Focus Knob: of the two knobs on the side of a microscope, it is the largest. It is used to focus on the specimen; it may move either the stage or the upper part of the microscope (in a relative up and down motion). Always focus with the coarse knob first. Fine Focus Knob: it’s the smaller round knob on the side of the microscope used to fine- tune the focus of your specimen after using the coarse adjustment knob. Stage Y-Axis Travel Knob: allows travel of slide up & down. Stage X-Axis Travel Knob: allows travel of slide left & right. Field Lens & Field Diaphragm Ring: (the light source) can be a bulb or a mirror and is usually found near the base of the microscope shining up through the stage. 6. List the different types of computer software used and give the function of each. Motic Images Plus 2.0ML: used the camera attached to the microscope to capture still and video images. ImageJ: analyzed the images captured. The colored images are turned to 8-bit grayscale images, the line tool is dragged horizontally across the cells, and a Plot Profile graph is made showing the gray level values that range from 0(black)-255(white) (with greys in between 0-255.) Excel: the values collected are entered in the form of XY scatter plots with the Y-axis representing the gray values and the X-axis representing the pixel numbers. Powerpoint: made slideshow with all of the data collected. 7. List the types of cells imaged and the characteristics of each. Amoeba: have pseudopods (false feet) and contractile vacuoles; cytoplasmic streaming is where there is a rush of the cytoplasm moving towards the pseudopods, making them extend outwards and move the amoeba around. Euglena: a protist with flagella at the anterior (front) end, a red eye spot, and natural chlorophyll (green) pigment coloration; is fast so one must focus on one that is spinning in a circle (or dying.) 8. What question(s) were being investigated in this lab Comparing the data and images: Which type of optics produced the most detailed image of the cells How did the line graphs indicate which image had the most detail visible LAB 2: Epifluorescence of Food Vacuoles 9. Describe the additional parts a light microscope must have to be used for epifluorescence imaging. Mercury Lamp Cooled CCD Camera Special Housing: excitation filter, cut-off/emission filter, and beam splitter. 10. Why did we use a cooled CCD camera to take images of epifluorescence Cooled CCD Camera: provide maximum sensitivity (with low background noise ((extra readings not needed)) to view relatively dim fluorescent dyes that fade during prolonged illumination prohibiting exposure of more than a few seconds. 11. What does a fluorochrome do specifically Fluorochrome: are fluorescent dyes that are excited by light of particular wavelengths and then emit light of different wavelengths in which are conjugated to another molecule that attaches to a particular molecule/organelle within the cell involved in the process of interest. 12. Which fluorochrome was used To what does it attach Tetramethylrhodamine-conjugated dextran: attaches/sticks to bacteria which serve as a food source for the paramecia to study the amount of food vacuoles after certain incubation times. 13. Draw the excitation and emission curves for this fluorochrome. The Fluorescence Excitation goes from 450-600 in wavelength (nm) while the Fluorescence Emission goes from 550-700 in wavelength (nm.) 14. What type of organism is a paramecium Paramecium: a single-celled eukaryotic ciliate that are large enough to visualize dynamic intracellular processes by using a microscope; carries out osmoregulation and feeds on microorganisms such as bacteria, algae, and yeasts. 15. Draw and label a diagram of a paramecium. *The “gullet” is also a “pharynx” **There is an “anal pore” at the anterior end for expulsion of waste/leftover food vacuoles. 16. Describe how the paramecium moves, eats and expels water. Moves by: cilia that cover the body to allow the cell to move with a synchronous motion. Eats by: an oral groove containing compound oral cilia that is used to draw/sweep food inside, then into the pharynx/gullet, then (when accumulated enough) breaks away and forms a food vacuole, then enzymes from the cytoplasm digest it all along the cell getting smaller and smaller, and lastly reaches the anal pore where the remains/undigested waste is expelled/removed. Expels Water by: osmoregulation carried out by a pair of contractile vacuoles which actively expel/pump out water absorbed by osmosis from its surroundings (hypotonic freshwater environment, which has a lower salt content than paramecia) to form an isotonic environment. 17. What was the purpose of the 4% paraformaldehyde solution 4% Paraformaldehyde solution: we added 550 microliters of 8% to the tube of 4% which doubled the volume and reduced the concentration of the tube to half. 4% will fix (kill and preserve) the paramecia to prevent further ingestion (of the bacteria) and make them easier to image. 18. What was the question being investigated in this experiment Based on the results: what can you conclude about the vacuole formation over time in paramecia 19. How were data collected and statistically analyzed Atleast 10 paramecia were taken up by a pipette and placed in a micro-centrifuge tube in which the fluorescent dextran solution was applied to. They were incubated (in a drawer) at different time periods and then fixed with the paraformaldehyde. They were then spun causing the paramecia to sink to the bottom of the tubes, made on a wet mount slide, and looked at under the epifluorescent microscope to count how many food vacuoles were there. The data were entered into a spreadsheet software (Statistica) where the independent variable/column was the group numbers/time intervals done and the dependent variable/column was the amount of food vacuoles seen. (ANOVA) performed a one-way ANOVA (determining differences between the groups) on the data to find the p- value for the post-hoc analysis (TUKEY’s) which gives <0.5 to have a significance. 20. What was your conclusion based on the analysis As the more time went on, it seemed that more food vacuoles were made until 70 minutes which was the best incubation time for the most food vacuoles and then the vacuoles decreased after that point. LAB 3: Protein Extraction from Muscle 21. Define proteome and proteomics. Proteome: the collection of proteins that comprise a cell, tissue, or an organism; differ from cell-to-cell, tissue-to-tissue, and organism-to-organism. Proteomics: the study of the function, structure, and interaction of proteins. 22. Describe myosin proteins in general and myosin II in particular. Myosin Proteins: important proteins in eukaryotic cells that function as molecular motors that convert chemical energy from ATP to mechanical energy thus generating force and movement. Myosin II: the conventional myosin involved in the contraction of animal muscle cells and in cytokinesis in non-muscle cells; very large protein consisting of 2 identical heavy chains and 2 pairs of light chains. 23. Draw and label a myosin II molecule. 24. Give a function of each part labeled (in 23.) Light chains: wrap around the neck of each head region and may be phosphorylated to regulate the activity of the heavy chains. Heavy chains: Head region: contains the catalytic and actin-binding site. Catalytic site: binds ATP (head has low affinity for actin.) ATP is hydrolyzed to ADP+P which causes the neck region to “flex” so the head is “cocked” (head has a high affinity for actin.) The neck then returns to its original state pulling on the actin filaments. 25. Explain how a sarcomere contracts. Sarcomere: contractile units made of myosin proteins and actin filaments in which many myosin proteins are bundled together such that the tails occur in the middle of the myosin bundle (thick filaments) and oppositely directed actin filaments/heads (thin filaments) occur near both ends; when both interact (the myosin acts on actin,) the thin filaments slide towards each other causing the sarcomere to contract. 26. Draw a figure that shows the relationship between sarcomeres, myofibrils, and muscle fibers (cells), and skeletal muscles. 27. How were the proteins extracted and denatured from fish muscle (be specific as to what each thing does) Laemmli (lysis) sample buffer to break down tissue, SDS to disrupt the lipid bilayer and coat the proteins with a negative charge, DTT to break disulfide bridges and stop protein-to-protein interactions, and Heat (90deg.) to break any lasting weak bonds. Were added to one fliptop tube, a piece of fish muscle was obtained and cut to 0.25cmX0.25cmX0.25cm and placed into a fliptop tube, was flicked 15 times to mechanically disrupt the tissue, incubated for 5 mins to give time for the buffer components to extract, solubilize, and partially denature the proteins, pour the buffer with extracted proteins but not the fish muscle itself into a new tube, heat the tube in a float for 10 mins at 90C, let tube cool, and freeze at -20C. 28. Why did we extract muscle proteins from 2 different types of fish To compare the sizes in the molecular weight of the MLC’s between the two and to find out if the Tilapia and Mahi Mahi had a common ancestral/evolutionary relationship. LAB 4: SDS PAGE and Western Blotting 29. Approximately how many different muscle specific denatured proteins were in your fish samples About 19 different muscle specific denatured proteins were in our fish samples. 30. What is SDS PAGE Why is it used Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis: used to separate denatured proteins from a mixture according to their molecular weight using an electric current to separate the proteins coated in SDS-containing sample buffer in a sieving gel matrix that separates the polypeptides by their size. 31. List the components needed to run SDS PAGE and give a function of each. Polyacrylamide Gel: positioned in a buffer-filled chamber between 2 electrodes and protein mixtures are loaded into wells at the top of the gel. Sieving matrix. Power Supply: generates a voltage gradient from negative to positive down the gel. PAGE Rigs including glass plates (10 x 20 cm), spacers, comb, and clamps: holds gel cassette in place for test. Protein sample: negatively charged so that it migrates through the gel toward the positively charged anode (larger moving slower than the smaller.) Bio-Rad Laemmli Sample Buffer (contains SDS): coats proteins in a negative charge so they move towards the positive anode. Kaleidoscope protein standards: help to determine migration of proteins. Controls: myosin and actin to determine if anything goes wrong. Running buffer: contains TrisGlycine-SDS is a pH buffer that provides salt ions and coats with negative charge. 32. What was the purpose of running stained standards on the gel Kaleidoscope Protein Standards: pre-stained standards that are genetically engineered proteins that have dyes covalently bound to them in which resolve into multicolored bands that move down the gel during electrophoresis and are then transferred from the flimsy membrane during the blotting procedure. Used to monitor the progress of polypeptide migration during the SDS-PAGE To determine the success of the blotting procedure To estimate the molecular weights of the polypeptides on the western blot. Standards act as a reference while the controls tell us whether or not our experiment worked properly. 33. How did the rabbit actin and myosin sample serve as both a positive and negative control Rabbit Myosin LC’s: should be labeled and act as the positive control. Rabbit Actin: should not be labeled and act as the negative control. 34. How did you know when to stop the electrophoresis When the blue tracking dye reaches the bottom of the gel. 35. What is Western blotting Why is it used Western Blotting: able to identify specific proteins from a complex mixture of proteins extracted from cells. Uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. 36. Draw, label, and give functions for the parts of the blotting sandwich. What caused the proteins to move LAB 5: Immunodetection of Myosin LCs 37. What is immunodetection Give examples of when immunodetection is used. Immunodetection: involves the use of antibodies to identify specific proteins or their subunits. Used when: diagnosing diseases (such as HIV or Mad Cow Disease) 38. What are primary antibodies How are they made Primary antibodies: what is produced in response to an antigen. Take a known antigen, inject it into one specimen, and the accumulated antibodies in the blood serum produced from that process are collected. 39. What are secondary antibodies How are they made Secondary antibodies: one specimen’s antibodies are injected into another specimen which is then collected and purified and eventually conjugated to horseradish peroxidase (HRP.) Recognize and bind to primary antibodies. 40. What purpose was served by HRP Where was it located Horseradish Peroxidase (HRP): dyes/conjugates to the secondary antibodies; enzyme catalyzes the oxidation of a substrate (4CN) in the presence of Hydrogen Peroxide that produces color (a purple/grey) so that the protein can be identified. 41. How does using a secondary antibody help in detecting small amounts of antigen By binding to primary antibodies. 42. What purpose was served by the blocker milk solution Blocker Milk Solution (5% milk and PBST Buffer): prevents non-specific binding. 43. Draw a diagram using symbols to show all the different protein interactions required to produce a band on your blot. 44. How did you collect data from your blot to make a standard curve Using PhotoStudio5 (allows you to view live images and capture still images), estimate the size of the myosin light chains (by using ImageJ) from the image taken (place cross in center of heaviest standard, read the y-coordinate and repeat with the next and next and so on heaviest standards, then the immunopositive bands, then construct a curve (usin Excel) and use log B. 10 function with all the standards, then make an XY scatter plot (with Y-axis as the log B. 10 masses and the X-axis as the pixels). 45. What 2 methods did you use to estimate the MW of your immunopositive bands Which is more accurate Estimated by eye: based on how far they traveled Use the trendline eqn. in excel (distance migrated ((Y coordinates)) and solve for Y ((more accurate.)) 46. What does R value indicate The equation and correlation coefficient for the line drawn straight through the data points on the scatter plot. Want to have R2 close to 1. 47. What exactly did you plot for your standard curve Why They have a linear relationship using Log B.10 of the molecular weight. (Weight determines how far the protein will travel.) 48. Draw and label a representative standard curve for your western blot. 49. How did you convert your estimate into kD Took the inverse log of the masses recorded.