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Compare and contrast brightfield, darkfield, and phase-contrast optics.
- Bright field- background is brightly lit and only colored specimens that absorb light at particular wavelengths and reflects or refracts other wavelengths that produce any contrast. Best for prepared slides of dead stained cells or live cells that have naturally occurring pigments.
- Phase contrast- a special condenser coupled with special objectives to produce a dimly lit background while allowing light refracted by structures in a transparent specimen to stand out as white (in phase) or black (out of phase) against the gray background. Can view live cells without pigment.
- Dark field- a disk is placed in the condenser so that only the outer edge of the light source is allowed to reach the specimen instead of light coming up through the specimen. Only the light that is reflected or refracted by the specimen into the objective will be visible. (a hollow cone of light. Can only see light that is highly refracted). Can view live cells without pigment.
Explain how quantitative data were collected from images.
We used image J to convert the images into grayscale. The line test gave numerical values across the image of gray values of the pixels that can be objectively analyzed
Draw and label representative graphs of line plots of gray values from images of cells taken with different optics.
Explain how line plots of gray values show contrast in images.
Changes in Gray values show the contrast in color. Shows numerically which optics shows the highest contrast in color of the specimen. More contrast means that the specimen is more visible.
Identify the parts of the light microscope and give a function of each.
- Eyepiece- used to view the specimen. Light comes up through here.
- Specimen holder- holds the specimen onto the stage
- Stage- where the specimen is held
- Condenser focus knob- raises and lowers the condenser
- Condenser aperture scale- allows you to choose between phase contrast and bright field and dark field by adjusting the angle of light. Focuses the light onto the specimen.
- Fine focus knob- makes small adjustments in the height of the stage. Used in high magnifications.
- Coarse adjustment knob- makes bigger adjustments to the height of the stage. Used in 4x magnification
- Binocular eyepiece tube- adjusts focus of the eyepiece
- Objective- varies the magnification. 4x, 10x, and 40x. (40, 100, 400).
- Interpupillary distance scale- changes the distance between the oculars
- Stage X and Y axis travel knob- moves the stage forward and backward
- Diopter adjustment ring- ??
List the different types of computer software used and give the function of each.
- Photostudio 5- allows you to view the live image as well as acquire still images
- Videoimpression 1.6- allows you to take videos of the images
- Image J- allows you to edit the pictures. In our case we change them to 8-bit (grayscale). Draw a line across and will show you the change in grayscale (gray level values). Allows you to produce quantitative and objective data.
- Excel- allows you to use values to make a graphPowerpoint- allows you put all the information together in a presentation
List the types of cells imaged and the characteristics of each.
- Cheek cell- eukaryotic, no natural pigments, motionless, mainly dead
- Amoeba- cytoplasmic streaming, psuedopods, no pigment, protist
- Euglena- flagellated, natural green pigment, has an outer pellicle to prevent squishing, protist
What question was being investigated in this lab?
- Which optics works best with which organism?
- How do you collect quantitative data?
Describe the additional parts a light microscope must have to be used for epifluorescence imaging
- Excitation filter- selects the color of light. Filter transmits light (lets it go through) or absorbs(stops). Special filter in the light that separates the wavelength needed to excite fluorescence from the total emission of the mercury lamp.
- Housing with beam splitter- directs the light from the lamp to the objectives and from the objectives to the oculars
- Cut-off filter- will absorb the excitation light scattered in the microscope but transmits the emitted light to the oculars and camera
- Mercury lamp- excites the fluorochrome by illuminating it from above through the objective
- Fluorochrome- gives off (emits) light. Fluorescent dye that is excited by a particular wavelength of light and emits a different wavelength of light
- MaxmDL- capture and process images and collects data
- Cooled CCD camera- provides maximum sensitivity with low background noise. More sensitive to light
Why did we use a cooled CCD camera to take images of epifluorescence?
It provides maximum sensitivity, with low background noise. More sensitive to light.
What does a fluorochrome do specifically?
They are fluorescent dyes that are excited by light of particular wavelengths and then emit light of different wavelengths
Which fluorochrome was used? To what does it attach?
Tetramethylrhodamine conjugated dextran(covalently bonded to the dextran). Rhodamine dyes are excited by wavelengths corresponding to green light and emit wavelengths corresponding to red light. The dextran will stick to the bacteria which serve as a food source for the paramecia
Draw the excitation and emission curves for this fluorochrome.
What type of organism is a paramecium?
Single-celled eukaryotic ciliates. Protist, predator, large
Draw and label a diagram of a paramecium.
Describe how the paramecium moves, eats and expels water.
Movement is made by synchronous movement of the cilia. Oral groove contains cilia which make a current of water (by beating) to pull their food in (bacteria). goes to pharynx. Forms food vacuole. As it moves through the cytoplasm, enzymes break it down. Digested food goes into cytoplasm and vacuole gets smaller. When vacuole reaches the anal groove (cytopyge) the remaining undigested food is expelled. Osmoregulation is carried out by a pair of contractile vacuoles which expel water by osmosis. Micronucleus shares information with another paramecium during conjugation.
What was the purpose of the methylcellulose solution?
The methylcellulose slows the paramecium down so that it is easier to count their vacuoles.
What was the question being investigated in this experiment?
Which time interval was optimal for food vacuole formation in paramecia?
How were data collected and statistically analyzed?
Each group incubated their specimens for a certain time interval. Vacuoles were counted for each group. The data was entered into a spreadsheet using statistica software. We then performed a one-way anova (one way because we have a single independent variable. Compares values overall and tells if it is statistically different but doesn’t say which are different). Food vacuole numbers were the dependent variable, time interval was the independent variable. The post-hoc analysis compares the means across all treatments. If the p values are <0.05 then the treatments are statistically different from one another
What was your conclusion based on the analysis?
Group 6 is the only group that is statistically different from 1 and 2. Because of this, it is statistically different. 90 minute was optimal because the p value was less that 0.05.
Define proteome and proteomics.
- Proteomics- the study of the function, structure, and interaction
- 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
Describe myosin proteins in general and myosin II in particular.
Myosin proteins are important proteins in eukaryotic cells. Myosin proteins function as molecular motors that convert chemical energy from ATP to mechanical energy thus generating force and movement. Myosin II is the conventional myosin involved in contraction of animal muscle cells and in cytokinesis in non-muscle cells. Myosin II is a very large protein consisting of 2 identical heavy chains and 2 pairs of light chains. The heavy chains have a long tail, a neck, and a globular head region.
Draw and label a myosin II molecule.
Give a function of each part labeled in 23.
- Myosin heavy- long tails wind around eachother. ~200 to 250 kD
- Head region- contains a catalytic site and an actin binding site. When the catalytic site binds ATP, the head has a low affinity for actin. ATP is hydrolyzed into ADP + P which causes the neck region to return to its original position pulling on the actin filaments.
- Tails- form coiled coils. When many exist (with actin) they form a sarcomere or contractile unit.
- Myosin light- wrap around the neck of heavy chains. They may be phosphorylated to regulate the heavy chains activity. ~15 to 25 kD.Neck- allowed to cock (bend)
Explain how a sarcomere contracts.
Sarcomere- contains myosin proteins and actin filaments arranges into contractile units. Myosin tails occurring in the middle while oppositely directed heads occur near both ends of the bundle. The myosin bundles (thick filaments) alternate with oppositely directed actin filaments (thin filaments). When the myosin heads interact with the actin filaments, the thin filaments slide towards each other causing the sarcomere to contract.
Draw a figure that shows the relationship between sarcomeres, myofibrils, and muscle fibers (cells), and skeletal muscles.
refer to lab sheet
How were the proteins extracted and denatured from fish muscle? (be specific as to what each thing does)
The tissue must first be broken down to release proteins from within the cells. The lipid bilayer must then be disrupted for the cells to release proteins and the proteins must be denatured to form linear polypeptides that will pass through the pores of the gel. The lysis sample buffer (laemmli) used to break open the muscle cells contain the ionic detergent sodium dodecyl sulfate (SDS)(is a detergent so solubilizes the membranes by getting in between lipid bilayer molecules) and a strong reducing agent dithiothrietol (DTT)> SDS coats the proteins in a negative charge and DTT breaks disulfide bridges as well as prevents protein to protein interactions. These substances interfere with the bonds in proteins that are necessary to maintain secondary, tertiary, and quaternary structures. Heating the ample to 90 degrees Celsius increases the kinetic energy of the polypeptides and further disrupts weak interactions to completely denature the proteins into linear polypeptide arrays
Why did we extract muscle proteins from 2 different types of fish?
To show that myosin is the same in different species. To show similarities of the fish myosin, and also compare them to rabbit myosin. Isn’t a difference between molecular weights of fish.
Approximately how many different muscle specific denatured proteins were in your fish samples?
19 possibilities in chart on lab handout. Only myosin light showed up on blot.
What is SDS PAGE? Why is it used?
SDS polyacrylamide gel electrophoresis is used to separate denatured protein form a mixture according to their molecular weight. Using an electric current, proteins coated in SDS contain sample buffer are separated in a sieving gel matrix that separated polypeptides by their size. A polyacrylamide gel is positioned in a buffer filled chamber between 2 electrodes and proteins mixtures are loaded into wells at the top of the gel. The electrodes are connected to a power supply that generates a voltage gradient from negative to positive down the gel. The SDS negatively charged proteins will migrate through the gel toward the positively charged anode with the larger polypeptides migrating more slowly that the smaller ones.
List the components needed to run SDS PAGE and give a function of each.
- SDS- sodium dodecyl sulfate- denatures proteins and emparts a negative charge
- Sieving gel matrix- separates polypeptides by their size. Proteins travel through
- Polyacrylamide gel- electrophilic gel.
- Electrodes- apply + and – charges so that – charged proteins migrate toward the positive end. Power supply 200 volts, voltage gradient.
- Wells- hold samplesBuffer- allows charges to run electrolyte solution. Keeps pH constant
What % gel did we use? Why was that specific % appropriate?
15% tris gel. To keep pH within a constant range. Also to allow only certain weights to flow through. The lower the percentage of the gel, the bigger the pores. The higher the percentage of the gel, the smaller the pores.
What was the purpose of running stained standards on the gel?
They are used to monitor the progress of polypeptide migration during SDS page and to determine the success of the blotting procedure. Also used to estimate the molecular weights of the polypeptides on the western blot.
How did the rabbit actin and myosin sample serve as both a positive and negative control?
- Positive control- the myosin standard shows what you should get if myosin is present in samples.
- If you see a band in the myosin, you know that the immunodetection worked, the gel ran okay, and the blotting worked.
- If no band is there, you know that something went wrong. Loading didn’t go properly?
- Negative control- the actin standard shows what you shouldn’t getIf actin shows up, something was wrong with the antibodies.
How did you know when to stop the electrophoresis?
When the blue tracking due reaches the bottom of the gel
What is Western blotting? Why is it used?
When polypeptides are transferred from within the surface of the fragile gel to the surface of a membrane made of nitrocellulose. It is used because the nitrocellulose membrane is more stable and longer lasting than a gel and polypeptides bound to it are more accessible to antibodies used in immunodetection. Binds protein well
Draw, label, and give functions for the parts of the blotting sandwich. What caused the proteins to move?
- Wick- acts to pull buffer from the reservoir and conduct the buffer to the blotting paper.
- Buffer- as it moves through the gel and the membrane, it will carry the proteins with it. Moves up the sandwich through capillary action and carries the proteins from the gel to the nitrocellulose membrane.
- Blotting paper- absorbs the buffer
- Plastic barrier- prevents the buffer from soaking thought the edges of the blotting paper that surround the gels. Makes it go through the gel instead of up around it.
- Paper towels- collects excess buffer. Provides continuous capillary action.
- 1kg weight- prevents sandwich from falling and makes sure there is good contact.
- Wrap- prevents evaporation
- Platform- raises the gels above the buffer reservoir so it won’t come in direct contact with the buffer reservoir
What is immunodetection? Give examples of when immunodetection is used.
The use of antibodies t0 identify specific proteins or subunits. Used in bioscience research, diagnostic tests, and medical therapies. Used when there is an invasion of foreign substances in the body.
What are primary antibodies? How are they made?
Primary antibodies- when exposed to a foreign antigen , most animals generate antibodies to that antigen. Over time, they will accumulate in the blood serum. There are made when an animal is injected with an antigen. They will produce antibodies in response.
What are secondary antibodies? How are they made?
Secondary antibodies are antibodies that recognize and bind to primary antibodies. They are produced by injecting primary antibodies into another species so that the foreign antibody will provoke an immune response. Primary antibody is seen as an invader.
What purpose was served by HRP? Where was it located?
Horse radish peroxidase is an enzyme that catalyzes the oxidation of a substrate that produces color so the protein can be identified. It was attached (covalently bonded) to the secondary antibody.
How does using a secondary antibody help in detecting small amounts of antigen?
Once purified, the secondary antibodies are tagged with a fluorescent dye or enzyme that produces a precipitate (makes it colorful so you can see it). Secondary antibody is specific for primary. Multiple secondary antibodies bind to each primary antibody. Provides more binding sites for the dye.
What purpose was served by the blocker milk solution?
It blocks non-specific binding sites on the nitrocellulose paper. So it does not bind to other proteins.
Draw a diagram using symbols to show all the different protein interactions required to produce a band on your blot.
refer to lab sheet
How did you collect data from your blot to make a standard curve?
- We took a picture of the western blot, and opened it in Image J. we were able to determine the Y coordinates (distances traveled) by each of the bands.
- Heena’s answer: collected data by taking a picture of the western blot and opening it up in a program called ImageJ. From there the Y coordinates of all bands in lane 2 and the heaviest bands in lanes 4-9. Then we followed the following instructions: open excel from the desktop. Type in the mass of each standard in one column and its corresponding Y coordinate (distance migrates) in the other column. A linear relationship exists between the migration of the proteins and the log10 of their molecular masses. In a third column you will record the log10 of the molecular masses. Click on the first cell in the third colum. Click the arrow beside the [s, then type log10 for the search term. Choose log10 and then click on the molecular mass of 250 kD. The computer will insert log10 of 250 in the first cell of the third column. Now click the second cell of the third column and insert log10 of 150 kD. Repeat the process with all the standards. Once you finish, make a graph of your data. Click insert, select chart, and then select XY scatter plot. Choos the second plot type (data points connected by smooth lines). Plot the log10 masses on the y-axis versus the distances traveled (in pixels, Y coordinates) on the x-axis. Include major and minor gridlines for each axis. Title and label the x and y axes on your graph. Why did we convert to log10 from kD?—there is a linear relationship between the log10 mass and the distance traveled. You need a linear relationship for the standard curve.
What 2 methods did you use to estimate the MW of your immunopositive bands? Which is more accurate?
We hand plotted the points and estimated the log10 mass by drawing a straight line from the point to the y-axis of the log10 mass values. We also used the strait line graph made from the known standards. We located their y axis location. Using the equation is most accurate.
What does R2 value indicate?
The r2 value shows how precise each estimate will be to the real value. The closer to 1.00, the better the value is. (the value is the estimate of the size of myosin light chains). It’s a correlation coefficient.
What exactly did you plot for your standard curve? Why?
- The standard cure showed the distance traveled of myosin light chains vs. the molecular weight.
- We plotted the “y axis location” of the known standards on the x-axis and the log10 of their known molecular weights on the y axis to create a line that would allow is to estimate the molecular masses of the myosin light chains.
Draw and label a representative standard curve for your western blot.
How did you convert your estimate into kD?
Converted estimate into kD by using a formula derived from trendline in excel. Y=Ax +B