asdf

Question 1

Define what is meant by an enzyme’s optimal temperature

Answer 1

range in which it performs its work most efficiently.

Question 2

Define what is meant by an enzyme’s optimal pH in your own words and explain why extreme pH’s
denature most enzymes

Answer 2

Radical alterations in pH can denature proteins. Enzymes are proteins. (unless they are RNA).

If one defines (e.g.) acidity as propensity to donate H+, a strong acid can’t make for a very electrostatically (?) neutral environment and, as described in the first response, charges are of central importance to protein folding. Hydrogen bonds would be liable to break, and, as the protein lost its shape, the most efficient arrangement of the molecules would be something else.

but perhaps there’s an intermediate between proper folding and denaturation in which the enzyme is beginning to lose its structure but its active site remains intact. The intermediate phase would usually require more energy so is not optimal.

Optimal pH would be the range in which the enzyme functions most efficiently, which would be related to the environment(s) anticipated in its design.

Question 3

Did all cell types tested contain mitochondria? Lysosomes? A Nucleus? Why or why not?

Answer 3

nearly all eukaryotes contain mitochondria

Prokaryotic cells do not contain mitochondria, nucleus or any other membrane-bound organelles

Plant cells have no lysosomes; instead they have vacuoles,

all eukaryotes contain a nucleus

Question 4

Why was 0.9% saline used for the cheek cells?

Answer 4

isotonic

meaning

it exerts the same osmotic pressure as the human intravascular environment

Question 5

Did the chloroplasts need to be stained with a vital dye to see them? Why or why not?

Answer 5

no

they contain chlorophyll whose light-absorbing pigments are green

Question 6

give more info (Neutr Red)

Answer 6

Neutral red is a pH indicator stain which is mainly absorbed by lysosomes, which are quite acidic, and vacuoles, which perform a similar function to lysosomes but exist in plant cells. Acidic bodies turn red in this stain, but if the pH becomes basic (Mycobacterium tuberculosis has evolved an enzyme which increases lysosomal pH, e.g.) the indicator stain turns yellow.

Question 7

janus green stains

Answer 7

mitochondria

Question 8

Do any particles move back to their original side of the membrane? Why or why not?

Answer 8

Of course; the pores are symmetrically permeable on either side of the membrane.

Question 9

Which molecule, O2 or CO2, diffuses across the cell membrane?

Answer 9

both

Question 10

The rising portion of the graph represents the depolarization of the neuron. Which channels are active during
this portion of the graph, and what happens to the charge of the neuron at that location?

Answer 10

Sodium-gated channels are active. Potassium-gated channels are becoming active but they take a moment to open (Na & K leak channels do not close)
The charge across the membrane, until now firmly negative inside the membrane and positive outside, begins to change, positive charge falling and negative charge rising—at the apex, the positive charge built up outside of the membrane becomes briefly negative, and the negative charge within becomes briefly positive.

Question 11

methylene blue stains what?

whY?

Answer 11

DNA and RNA

it’s attracted to their negative charges

Question 12

The falling portion of the graph represents the repolarization of the neuron. Which channels are active during
this portion of the graph, and what happens to the charge of the neuron at that location?

Answer 12

It appears that while Na-gated channels are still technically active, a permanently-attached Na molecule is blocking the Na-gated channels. So sodium stops pouring in approximately when the charge is neutral and falling. K-gated channels which opened more slowly stay open longer, and potassium continues to rush out. The transmembrane charge reverses again, inside the membrane which was briefly positive it falls to neutral then returns to a negative charge which builds awhile so the neurone cannot fire again right away

Question 13

What happens to the concentrations of Na+ and K+ (both inside the cell and outside the cell) during an action
potential?

Answer 13

Sodium rushes in and potassium rushes out

Question 14

Note the charges inside the cell and outside the cell. What happens to these charges when the action potential
arrives?

Answer 14

they briefly switch. built up negative charge within becomes positive, built up positive charge outside becomes negative

Question 15

Note the voltage inside the cell at rest (the flat portion of the graph). How do you think the cell maintains that
resting voltage?

Answer 15

Through constant use of the sodium-potassium pump

Na and K have same charge

3 Na out
2 K in

Question 16

Note the voltage inside the cell at rest (the flat portion of the graph). How do you think the cell maintains that
resting voltage?

Answer 16

Through constant use of the sodium-potassium pump

Na and K have same charge

3 Na out
2 K in

Question 17

what do all three types of ELISA have in common

Answer 17

pos and neg controls

Question 18

name 3 types of elisa

Answer 18

direct
indirect
sandwich

Question 19

describe the basic ELISA setup – what is done beforehand

Answer 19

beforehand: an antibody and enzyme are spliced together or ‘conjugated’. (The antibody’s target antigen is the substance being measured by the test — the enzyme’s target substrate creates a reaction, often colour change.)

Question 20

ELISA steps?

Answer 20

sample

rinse

antibody

rinse

substrate

Question 21

what’s diff about indirect ELISA

Answer 21

Antibody 2 is the conjugate in this model; it will react with the substrate and act as an indicator.

Question 22

sandwich ELISA

Answer 22

similar to the indirect ELISA, but a capture antibody coats the plates or the bottoms of the wells before step 1 begins. The advantage here is that the capture antibody will decrease adsorption of extraneous proteins (e.g., ‘other stuff’ found in a sample; serum, sputum, et cetera) which have no bearing on the test.

Question 23

2. Based on the protocol you used, which type of ELISA was performed in lab? What is one
   advantage of this kind of ELISA?

Answer 23

indirect

It’s more definite than the direct ELISA. If the rule is that most substances will be washed away instead of adsorbing on to the bottoms of the wells, a more complex process requiring multiple steps will decrease the number of false positive results.

Question 24

ELISA: what if buffer wash were skipped?

Answer 24

all wells would give pos reading and test would be meaningless

Question 25

ELISA: why use wash?

Answer 25

The test is engineered in a certain way; to wit, we know beforehand that the antigen to which the first antibody binds is what we’re intending to measure. We know also that the conjugate enzyme will react with the final substrate. What is meaningful is whether or not all of these small structures get adsorbed on to the bottom of the well.

Question 26

A false negative is

Answer 26

a negative test result despite the presence of what was being tested for.

Question 27

A false positive is

Answer 27

a positive test result despite the absence of what was being tested for.

Question 28

What are negative and positive controls, and why are they so important to this type of
experiment?

Answer 28

positive controls should correctly indicate the presence of what is being tested for (indicated by a colour change, e.g.) — the negative controls should correctly indicate the absence of what is being tested for.

Positive and negative controls indicate the antigen is correctly identified by the test — that the correct substances are being used, the antibodies and other components are not too old to bind and react, et cetera. The two controls, functioning correctly, help one to trust the results (true negative) and they indicate nothing has gone wrong within the procedure (true positive).

Question 29

What are negative and positive controls, and why are they so important to this type of
experiment?

Answer 29

positive controls should correctly indicate the presence of what is being tested for (indicated by a colour change, e.g.) — the negative controls should correctly indicate the absence of what is being tested for.

Positive and negative controls indicate the antigen is correctly identified by the test — that the correct substances are being used, the antibodies and other components are not too old to bind and react, et cetera. The two controls, functioning correctly, help one to trust the results (true negative) and they indicate nothing has gone wrong within the procedure (true positive).

Question 30

Why is each sample added to three wells instead of just one?

Answer 30

Running each sample in three wells (in triplicate) gives a wider experimental set, within which erroneous results may be identified.

Question 31

6. Explain how the same concepts used in ELISA may be used in a COVID-19 antibody test.

Answer 31

More basically, any substance for which antibodies may conceivably be produced — antibodies to which some indicator which will have a reaction or give a result visible to the naked eye can be attached — in any such example, an ELISA or ELISA-like test could be developed.

Question 32

7. What are some potential limitations to ELISA testing? Use real-life examples in your answer.

Answer 32

not all enzymes / antibodies are sufficiently specific. They’re mostly specific, but there’s a possibility of cross-reactivity
• HIV or other immune system compromise — if antibodies are the tested substance, that’s because we rely on them being produced in an infected host. This doesn’t always happen.
• Occasionally an antigen will not bind properly to antibodies. As petri dish results can be affected by microbial contaminants, so (presumably) can ELISA be affected by chemical or biochemical contaminants which alter pH. Some of these may even be produced by the body in the presence of the target antigen — acidotic or alkalotic states

Question 33

What determines the resolution limit of a microscope?

Answer 33

the resolution limit of a microscope depends on several factors: the lenses used (and their quality) and the type of radiation passed through the specimen (white light, selected frequencies of light, electrons) are the most important.

The interaction between light and the sample is important too; the sample should be greater than or equal to about half of a wavelength of the radiation used to visualise it.

Question 34

Is the purpose of the fluorescence
microscope to increase the magnification beyond that of a typical light microscope? Why or why
not?

Answer 34

Fluorescence microscopy does not increase the magnification. It lights specific features by exciting fluorescing molecules; light passes through (several?) narrow apertures, and the light source can be a laser beam, which is amplified or ‘intensified’ if you will. All of these in concert should return the clearest image possible using visible light as a conveyance.

Question 35

2. What does immersion oil do and why?

Answer 35

When light passes through any medium, it changes direction (a vacuum is not a medium for these purposes, and ‘thin air’ is, in that atoms are floating around in gaseous phase.) So the wave properties of light are altered as they pass through air; this is refraction, which pertains specifically to velocity. Velocity as a physical concept has direction. If light changes direction after passing through the specimen and radiates into the environment, then the information it was carrying gets lost; the resolution of the resultant image will be compromised.

(If each photon is carrying information, then to lose a few would be like losing pixels on a computer screen — the image would become harder to read as it lost information.)

It seems that the mechanism of immersion oil’s focussing power relates to its refractive index: light passes through the oil more slowly and carries the information directly from the slide to the objective lens, which is immersed in the oil too. The light is bent into the microscope instead of obeying rules of ambiant scattering.

Use of immersion oil would therefore result in a clearer image.

Question 36

3. Compare and contrast the four types of fluorescence microscopy we discussed in lab:
   1) GFP fusion proteins 2) immunofluorescence – direct and indirect, 3) vital dyes such as
   mitotracker, and 4) phalloidin.

Answer 36

All four examples tag specific organic components.

The first two types (GFP and immunofluorescence) involve splicing an additional domain into existing proteins.

GFP fusion proteins — fluorescing AA domain is inserted at on end of the target protein (the protein we wish to track). So the target protein would glow, in this case, green. This method may have two major problems: GFP is a rather large domain, and GFP can agglutinate when present in a certain quantity; both of these mean that the target protein may behave unpredictably after GFP tagging.

Immunofluorescence — fluorescing AA domain insertion into antibodies. Direct immunofluorescence tags the antibody whose intended antigen is the target protein. Indirect immunofluorescence tags the antibody which targets the antibody whose intended antigen is the target protein. Indirect immunofluorescence turns out to be much more accurate than direct immunofluorescence. Immunofluorescence is probably better than tagging the target protein directly because it’s got a far lower chance of influencing its behaviour (observation distorting results). Disadvantages of immunofluorescence lie in the complexity of cultivating the antibodies, which requires significant time or money. Indirect immunofluorescence gets particularly expensive because the immune response to the first antibody is generated by inoculating the antibody into a larger organism, typically a larger mammal.

Vital dyes identify specific cellular components, particularly organelles. MitoTracker stains mitochondria whose membrane potentials result from activity in the Krebs cycle. It’s not clear to the students why mitochondria have a negative membrane potential when they collect protons in the inter-membrane space, but further research will be conducted (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792320/). For the purposes of this activity, MitoTracker is cationic, and the membrane potential of a mitochondrion, according to experts, is negative, so the dye is drawn to mitochondria specifically. It is unclear why it is not then drawn to the cell’s nucleus too. Vital dyes are the simplest (and likely cheaper by far since they do not require genetic modification) of the fluorescence microscopy techniques discussed, so if they serve the given purpose, they would be used first.

An actin is one of the group of proteins that construct microfilaments, the thinner parts of the cytoskeleton.

Phalloidin prevents actin from being broken down. It incorporates into the actin structure and allows visualisation of the cytoskeleton. In fluorescence microscopy, the phalloidin would probably contain fluorescent tags similar to those in fusion proteins, but with a particular affinity for cytoskeleton.

Question 37

4. How does a confocal microscope work, and how can it be used. How might it offer an advantage
   over some other types of microscopy?

Answer 37

A confocal microscope focuses light through two narrow apertures, creating a strong, direct beam. Using laser or amplified light, it sort of ‘reads’ the sample in 2D slices, making very thin cuts, and these slices can be combined to form a 3D image. It improves the resolution and may give additional insight into the 3D structure (while the sample is three dimensional, it is normally observed through a lens in 2 dimensions)

Question 38

5. Explain how the wavelengths of light are important and how they change when the technique is
   used (i.e. to see green, what color do we use?).

Answer 38

The wavelengths of light represent particle excitation; a shorter wavelength can discern somewhat smaller shapes (again, the sample should be greater than or equal to about half of a wavelength of the radiation used to visualise it; this is a physical principal). So higher energy light waves such as blues are ideal to convey more visual information. Some energy is lost in the form of heat which can cause the light to drop to a slightly lower frequency; thus blue drops to green, or green to red. Sometimes a higher-frequency blue drops to a lower-frequency blue. The original light excitation bounces off the matter and is transmitted to the observation device, but in fluorescence microscopy it must first pass through a dichroic mirror, which absorbs and reflects different wavelengths too.

Question 39

6. Explain what structures you saw with fluorescence microscopy in lab, and what type of cells we
   used.

Answer 39

We saw microfilaments of the cytoskeleton and mitochondria in bovine pulmonary arterial endothelia (BPAE). MitoTracker might have been used for the mitochondria slides, and phalloidin to stain the actin structures.

Question 40

7. Why is a protein’s location so important to a cell? Why is an organelles shape/structure so
   important to a cell?

Answer 40

Location determines function and ability to function (i.e., in the wrong place a protein may perform functions it wasn’t intended to perform; inversely, it cannot perform its intended function if it cannot physically bind to anything). At the cellular level, physical shape is the prime identifier; it determines fit, which determines protein interaction, and proteins form the infrastructure of the cell. Once a cell is functional, many processes presuppose the shape and structure of its organelles, again because messages are sent via physical binding and interaction which is regulated by physical shape.

Question 41

8. Explain how the information we gain using fluorescence microscopy could be applied to research?

Answer 41

It’s faster to explain how it could not be applied; it could not be applied to experiments no human ever imagines. Fluorescence microscopy is particularly useful for identifying components of cell structure. More information more readily available facilitates all related scientific research. This is particularly important as regards most cells and microbes, of which one of the prime difficulties in study comes from the simple fact that they are very small and hard to see.

The student did not get a chance to inspect the fluorescent microscope’s emissions mirrors, but suspects that it would be very useful in studying function, cell proliferation, etc., to be able to switch between high-resolution images of different structures when inspecting a sample.

Question 42

in your own words and explain what happens if

the enzyme solution is too cold or too hot.

Answer 42

Most functions of life slow at reduced temperatures; whereas applying heat will increase enzymatic activity until a threshold is crossed after which heat denatures the enzymes

(not all at once, though, because natural systems are seldom heated uniformly, so at high temperatures the activity will slow somewhat before rapidly dropping off to zero).

Question 43

enzyme no longer able to function – means what?

Answer 43

active site is compromised

Question 44

denaturation –

Answer 44

loss of nature – a protein would no longer be able to function