Embryology - Lab Final Flashcards
How large is the field of view in your microscope at 100X?
1800 micrometers
How large is the field of view in your microscope at 400X?
450 micrometers
Distinctive structural properties of a primordial follicle?
It is made up of squamous epithelial cells, located on the very edge of the ovary
Distinctive structural properties of the germinal epithelium?
It is the very outer layer of ovary itself, made up of oogonia
Distinctive structural properties of primary follicles?
They are made up of a single layer of cuboidal epithelial cells, there are more of them than in a primordial follicle, located more toward the middle of the ovary using primordial follicles as a reference point .
Distinctive structural properties of granulosa cells?
They are supporting cells which surround oocytes
Distinctive structural properties of the corpus luteum?
It is an area of lighter staining with a darker edge
Distinctive structural properties of a follicular antrum?
It is the space between granulosa cells and a primary oocyte
Distinctive structural properties of the cumulus oophorus?
It is the name given tot eh granulosa cells immediately attached to the primary oocyte when an antrum is present (as opposed to the granulosa cells that make up the follicle)
Distinctive structural properties of primary oocytes?
They are located withing primary and secondary follicles
Distinctive structural properties of secondary follicles?
Made up of multiple layers of cuboidal cells
Distinctive structural properties of the zona pellucida?
The dark pink tint surrounding a primary oocyte
What is a Graafian follicle?
a fully developed, mature follicle containing a primary oocyte as well as an antrum
What is a seminiferous tubule?
A hollow centered tubule containing developing sperm
Distinctive structural properties of spermatogonia?
Almost attached to the basement membrane of the tubule, they are the stem cells which will develop into sperm.
Distinctive structural properties of primary spermatocytes?
Found immediately above spermatogonia, the largest of the developing sperm stages
Distinctive structural properties secondary spermatocytes?
Smaller than primary spermatocytes, almost half the size
Distinctive structural properties spermatids?
The smallest of the developing sperm, they are found immediately before spermatozoa, they do not contain flagella
Distinctive structural properties of sertoli cells?
Look almost triangular and long, they extend from the BM up into the middle of the seminiferous tubules
Distinctive structural properties interstitial cells?
Found outside of the seminiferous tubules in triangular like clusters
Distinctive structural properties of spermatozoa?
Located in the intermost part of the tubule, the only developing sperm that contain flagella since at this point they are fully developed.
Distinctive structural properties of the basement membrane?
AKA basal lamina, surround the seminiferous tubules
a trillion or so immune cells are housed in organs such as the _________ and ___________ and periodically circle the bloodstream
spleen, lymph nodes
What is the immunilogical term for foreign proteins?
antigens
What do B lymphocytes do in response to the presence of antigens in the body?
They produce antibodies (also called immunoglobulins) which bind to the antigen causing the microorganism to be destroyed.
Aside from dealing with foreign organisms that could harm the body, what other important role does the immune system play?
Recognizing and destroying potential tumor cells in our bodies.
What was the purpose of the detection of yolk antibodies experiment?
In mammals, the vulnerable embryo is protected by the mother’s antibodies crossing the placenta and protecting the embryo. In this experiment we sought to determine whether or not chicken eggs have antibodies inserted into them for the protection of the embryo as well.
Blood serum from which animals is often used to obtain antibodies?
mice, rabbits, goats, and horses
What are some things that egg yolk is made up of?
stock-piled proteins, nucleotides, mRNAs, and lipids
If antibodies are present in an egg where should we expect to find them?
In the egg’s cytoplasm (yolk cytoplasm), not in yolk granules or any other organelles
How did we separate the yolk cytoplasm from the granules and organelles?
Via centrifugation.
How does a centrifuge work?
It pellets things using a combination of force and time (g-min)
What is the difference between an antibody and an antigen?
An antibody is produced by the hosts immune system and an antigen is a foreign protein found on an invading microorganism
What is the difference between rpm and xg?
RPM = how fast the centrifuge is spinning xg = the speed of the centrifuge x the radius of the object to the center of the centrifuge
Which immune system-stimulating proteins are found on the surface of microorganims?
antigens
In what units is the combination of time and centrifugal force expressed?
g-min
List 6 properties of proteins that can be used to purify them
- molecular weight
- net charge
- degree of hydrophobicity
- ability to bind to a certain substrate
- their polysaccharide chains (if any)
- differential solubility
Which protein property was exploited in order purify the antibodies from other cytoplasmic proteins using ammonium sulfate?
Differential solubility
How does ammonium sulfate precipitation of antibodies work?
When ammonium sulfate is added to the solution it breaks into ammonia and sulfate ions which readily try to bind to water. Eventually they bind to so many water molecules that the antibodies can no longer bind to the water, so instead they start binding to each other forming aggregates
At what percentage of ammonium sulfate saturation do antibodies precipitate?
at 50%
How are the antibodies purified once they are precipitated out of the cytoplasmic solution?
They can be redissolved in a smaller amount of water making them more concentrated and more pure.
On what 3 things does precipitation of proteins depend?
- concentration of ions present
- temperature
- pH
What is dialysis used for?
It is a way to separate low and high molecular weight substances.
Explain the process of dialysis
They solution containing the proteins desired for purification is placed inside of a bag that is then placed into water. The dialysis bag is permeable but only to molecules less than 10,000 da. Because of diffusion substances that are not desired such as ammonium and sulfate ions will readily diffuse through the semipermeable membrane of the bag leaving the desired proteins behind. This process takes several hours.
How do you concentrate proteins obtained via dialysis?
You simply wait for the water to evaporate directly out of the dialysis bag or a tube containing the dialysate
Give two reasons that a dialysis would ideally be performed in a cold room
- to prohibit the growth of bacteria
2. to prevent (slow) protease activiey
Give two reasons why it is necessary to maintain a neutral pH during the addition of ammonium sulfate to a solution
- so that the proteins do not precipitate based on pH
2. the proteins could denature if the pH is not neutral
When is it better to use a Lowry Assay and when is it better to use a Bradford Assay?
Lowry Assay = superior for solutions with high concentrations of proteins
Bradford = faster and more sensitive but mostly for low concentration solutions
What are two potential problems with using a Lowry Assay?
- the lowry assay is unable to accurately quantitate transmembrane proteins
- certain substances may interfere with the chemistry of the assay, altering the pH so that the chemical fails to react, or causing a precipitate to form
How do we fix the problem that Lowry assays cannot accurately measure amounts of transmembrane proteins?
By adding a low conc. of the detergent SDS which will dissolve vesicles and sheets of membranes to release these proteins
Will undissolved vessicle granules produce a false positive or a false negative spectrophotometric results?
False positive results
How do you read the results of a Lowry assay?
Using a spectrophotometer, which measures the optical density (no units) of a solution. The more intense the color of a sample, the more light is absorbed and therefore the higher its optical density and higher its concentration of proteins
Lowry assay results should be nearly linear up to concentrations of?
2 mg/mL
Spectrophotometer readings of optical density are only reliable up to a value of??
2.00 (normally, ours is 1.5)
If optical density is a relative value with no units how do we obtain an absolute value given an optical density reading?
By comparing a known standard to it
What are some characteristics of an ideal protein assay?
- low conc. allow for more accurate results
- an accurate standard to compare it to
- an OD reading of less than 2.00
- one that does not contain transmembrane proteins (or if it does they are dissolved)
- does not have interfering chemicals
Why is the importance of graphing data?
It helps to emphasize or illustrate a point that you want to make
How should data point values be presented?
As averages whenever possible
What is important to include when presenting graphs of data averages?
It is important to include error bars and include in the figure legend what type of error they indicate
When is it best to present data as a bar graph?
When the data is discontinuous
When is it best to present data as a line graph?
When it is continuous
When would you not connect data points?
When the data is qualitative and not continuous (e.g. blood type, gender, etc.)
Why do you draw a smooth line to approximately connect data points rather than connecting them literally?
To account for errors and erratic jumps in data points due to sampling
What is the independent variable? On which axis should it be plotted on?
It is the manipulated variable and should be plotted on the horizontal axis
What is the dependent variable? On which axis should it be plotted on?
It is the measured variable and should be plotted on the vertical axis.
How should the axes of a graph be labelled?
They should indicate the variable being measured as well as the units of measurement (if any)
Why should the scales of a graph almost always being at zero?
So as to not over-dramatize the results and differences between them
What are outliers? How should they be dealt with when presenting data?
They are nonsensible or bad data, they should be carefully and honestly discarded
Why should the most important comparisons in a set of data be presented on one graph?
So that it is easier to visualize and compare them
When can the word significant be used to describe similarites or differences in data?
‘significant’ can only be used only when statistical analysis has been used to support it
How should the names of organisms be presented?
Organism names are always italicized, with the genus capitalized but the species not.
Why is it useful to plot negative control values when presenting data?
Because they help to reveal the ease or difficulty with which values that were hypothesized or tested can be distinguished from control or background values
What should be in a figure legend?
The purpose of the graph, the conclusion of the graph and how the conclusion was arrived at as well as the value of N. Also mention the type of statistical analysis used and a P value
Why is calculating the percent change as opposed to the absolute change between variables useful?
Because it will account for individuals that have widely disparate start values
How do you calculate percent difference given to percentages?
Subtract initial percentage from the final percentage and divide all of that by the initial percentage
What does SDS-PAGE stand for?
Sodium dodecyl sulfate polyacrylaminde gel electrophoresis.
Why is gel electrophoresis so useful in molecular biological research?
Because it is a rapid why to find out not only the amount of proteins in a mixture but also their relative size bases on molecular weight
What is SDS?
sodium dodecyl sulfate, it is a negatively charged and stongly ionic detergent
What 4 critical roles does SDS play in gel electrophoresis?
- it dissolves membranes
- breaks hydrophobic bonds, separating multi-subunit proteins
- unfolds proteins by breaking hydrophobic and ionic bonds
- coats the proteins in a negative charge
How does gel electrophoresis work?
Proteins are coated in a negative charge and placed in a buffer that is on a polyacrylamide and the proteins are attracted to a positive charge on the other side of the well in which they were placed. Since they are unfolded and broken apart they will simply flow through the gel depending on their size. Larger proteins will migrate slower and smaller proteins will migrate faster and further down the gel
What 5 things can be found in the sample buffer used in gel electrophoresis
- SDS
- Tris
- Glycerol
- Mercaptoethanol
- Bromphenol blue dye
What problem could be presented when carrying out gel electrophoresis? And how do we get around that?
That the proteins at the bottom of the well would have a head start, and that could be fixed by forcing the proteins to stack up at the top of the gel (this can be accomplished by there being a large difference in pH and ionic strength between the resolving gel and the gel used to make the wells)
At what point should the two chains of antibodies be found at after gel electrophoresis has been done?
Light chain = 25,000 dalton
Heavy chain = 50,000 dalton
How do proteins get separated during electrophoresis?
By molecular weight through a polyacrylamide gel
Why are two gels needed (resolving gel and stacking gel) in gel electrophoresis?
In order to stack the initial proteins so that none of them have a head start by beginning at different placed in the well
Would having a very concentrated sample in a Lowry assay over or under estimate the protein conc?
It would underestimate it
How can we determine the exact molecular weight of a yolk protein on an SDS-PAGE?
By using known standards
What was the purpose of performing the ELISA experiment?
To determine what types of antibodies were present in the egg yolks
What does ELISA stand for?
Enzyme-linked Immunosorbent assay
How is an ELISA carried out?
Tween-20 is added in order to prevent non-specific binding of proteins than their antigens, then an antiglobulin is added. If both antigen and antibody are present than the antiglobulin will bind. A colorless substrate is added and the intensity of the color is proportional to the amount of antigen, antibody, antiglobulin and the amount of time the enzyme is allowed to react
How is an ELISA quantitated?
Using a microplate reader
What is a positive control?
It is used with the expectation of a positive in order to ensure that all enzymes and reagents are working possibly
What is a negative control?
It is used with the expectation that a negative result will arise, confirming that no outside factors will affect the results of the experiment
What result would you expect if the primary or secondary antibodies were not washed away in the ELISA?
You would get false positive results?
What is “background binding” in an ELISA?
It is when the antibodies attach to proteins other than the antigens despite the addition of tween 20
Why are zebrafish better to use than mice as cancer avatars?
- cheaper
2. faster
Why might zebrafish not be better cancer avatars than mice?
- they are more evolutionarily distant from humans
2. not all human drugs work in zebra fish
What are the 3 hallmarks of human solid tumors?
- rapid cell division
- ability to spread
- angiogenesis
How long would it take to test a drug for cancer in a fish avatar?
2-3 weeks
Why are zebrafish especially good models for studying embryonic development?
Because unlike mice or frogs you can rapidly and easily observe the fish as it develops within its traslucent egg outside of the mother
What is the key to zebrafish mutant search?
A strategy called “saturation mutagenesis” = treat adult males with a chemical mutagen and watch for abnormal development in offsprintg 3 generectumations
When was the zebrafish first considered as an option to use in studying development?
In the 1970’s
What is a retino-tectal connection?
A connection of the axons from the retina to the tectum in the brains visual center
What critical research piece are developmental biologists working with zebrafish missing?
They have yet to fully develop a genome map of zebrafish
