BSCI330 Lab Practical

Ligand

some extracellular signal

Substrate def.

target upon which an enzyme acts

Magnification

the ability of a lens to magnify, expressed as the ratio of the size of the image to that of the object

Solubility

the degree to which the protein dissolves in the surrounding solvent

What factors determine tertiary protein structure?

the non-covalent attractions among side chains (R groups)

Solvation

the process by which water molecules form hydration shells around folded proteins

What does the michaelis menten equation describe

the relationship between the rate of product formation by an enzyme to the concentration of the substrate

Resolution

the shortest distance between 2 points on a specimen that can still be distinguished by the observer as separate entities

When using Trypan blue dye.... if the cells appear blue (stained) what does this tell you about them?

they are dead

WST Assay: when colorimetric product is not formed, what does that say about the cells?

they are dead, dying, or metabolically inactive

When using Trypan blue dye.... if the cells appear clear and unstained what does this tell you about them?

they are healthy

WST Assay: when colorimetric product is formed, what does that say about the cells?

they are live, and metabolically active

What is the goal of protein precipitation methods?

to decrease the solubility of the protein in solution to get it to precipitate

What was the annexin V / propidium Iodide assay used for?

to look for plasma membrane changes associated with cell death

T or F: Cytochrome C makes up part of the apoptosome

true

What are some of the enzymatic changes that occur during apoptosis?

- activation of caspase enzymes - proteolytic cleavage of their substrates

What are some of the plasma membrane changes that occur during apoptosis?

- change in phospholipid distribution - late changes in permeability

Cardiac pacemaker cells are able to facilitate conduction because they .....

- have gap junctions - are capable of self-generated contractions - are connected by channels that allow the free passage of ions / small molecules

What are some of the similarities / differences between a microplate and a standard spectrophotometer?

- microplate measures vertically - microplate can do more than 1 reading at a time - both measure absorbance

What are some of the mitochondrial changes that occur during apoptosis?

- recruitment of specific proteins to mitochondrial membrane - release of cytochrome C - changes in mitochondrial membrane potential

How does both the sympathetic and parasympathetic nervous system effect HR?

- sym increases HR - para decreases HR

Why are enzymes considered catalysts?

- they greatly increase the rate of a specific rxn by decreasing the Ea - neither consumed nor modified by the rxn

What role does the detergent play in gel electrophoresis? What is the detergent that we used in our experiment?

- used SDS - binds to proteins proportionally to their masses (bigger mass binds more SDS) - unravels proteins --> SDS coats entire length of polypeptide chain --> polypeptide takes on a uniform rod shape (unified charge-mass ratio) allowing for their separation according to size only

What role does the stationary phase (matrix) play in gel electrophoresis? What is the matrix we used?

- used polyacrylamide - forms a porous matrix --> increases frictional coefficient --> larger molecules move slower through it --> separates molecules according to size (large at top of gel, small at bottom of gel) - pore size measured by gel percentage (4-20%) where the higher % gel, the smaller the pore size - can use single % or a gradient

Non-Competitive inhibition

- when the inhib. binds, the enzyme undergoes a conformation change, rendering it inactive - presence of the inhib. reduces the enzymes innate catalytic ability, regardless of [substrate] - reversible

A student makes three serial 1:5 dilutions of a 3 mg/mL BSA standard. Of the three samples she generates, what is the concentration of her most diluted sample? 0.6 mg/mL 0.024 mg/mL 0.12 mg/mL 0.003 mg/mL

0.024 mg/mL

How many reactions does a specific enzyme usually catalyze?

1

2 types of lymphoma

1) hogkins - manifests in lymphatic system 2) non-hogkins - manifests in blood

3 steps of cell communication

1) reception - binding of ligand + receptor 2) transduction - relay of signal through a cascade of intracellular molecules and their effector proteins 3) resposne - specific change in cell behavior carried out by cytoplasmic machinery targets of effector proteins

In a phase contrast microscope, the phase plate causes undiffracted light waves to undergo a phase shift by _______ wavelength. There is a phase shift of ___ caused by diffracted light. The total out of phase wavelength change is ____

1/4, 1/4, 1/2

The same data from question 3 above is shown. Again, assume that these two samples were each diuted by a factor of 10 prior to the absorbance reading, and the A595 values shown are averaged from triplicate samples. Ammonium Sulfate Soluble (AS): A595 = 0.54 mg/mL BSA standard curve extinction coefficient: 0.15 Pathlength = 1 Whole Plasma (WP): A595 = 0.92 Ammonium Sulfate Soluble (AS): A595 = 0.54 mg/mL BSA standard curve extinction coefficient: 0.15 Pathlength = 1 To generate her AS sample during her precipitation experiment, the student had added 0.2 mL of 100% ammonium sulfate to 0.2 mL of her WP sample. According to the above data, approximately how many mg of total protein were in this 0.2mL WP sample? about 7 mg about 1.2 mg about 12 mg about 0.3 mg

12

A student obtains the following data from a Bradford Assay she conducts on two protein samples obtained from an ammonium sulfate precipitation experiment. Note that these samples were each diuted by a factor of 10 prior to the absorbance reading. A595 values shown are averaged from triplicate samples. Ammonium Sulfate Soluble (AS): A595 = 0.54 mg/mL BSA standard curve extinction coefficient: 0.15 Pathlength = 1 According to this data, what is the approximate concentration of her AS sample? 0.28 mg/mL 28 mg/mL 3.6 mg/mL 36 mg/mL

36

If a polypeptide with the amino acid sequence below is subjected to trypsin enzyme-mediated hydrolysis, how many peptide fragments would be generated? MET-SER-ARG-TYR-THR-LYS-GLY-LEU-LEU-ARG-TYR-PHE-PHE-VAL NONE 2 3 4 5

4

A group of students test the basal and drug-induced heart rates of 5 Daphnia for the drug, lidocaine. For each Daphnia, they measure the basal and drug-induced heart rate 3 times. What is the sample size for their experiment? 3 10 5 15

5

In the 'Salting Out Method' protocol your group followed in Lab Exercise III, you precipitated plasma proteins in what FINAL concentration of ammonium sulfate? 25% 50% 75% 100%

50%

% composition of blood

55% plasma 45% formed elements <1% buffy coat

Beer Lambert law

A = ebc A = absorbance e = extinction coef. b = path length (usually 1) c = concentration

A given objective lens has a N.A. value of 0.45. Assuming a Lamda value of 550 nm, which of the following cellular structures/features could be resolved using this objective (choose all that apply)? A. a nucleus B. a lysosome C. diameter of a microfilament D. length of a mitochondrion (assume about 3 um)

A and D

Agonist drug

A drug that mimics the effect that the natural ligand has on its receptor - drugs that bear structural similarity to the receptors natural ligand - also called an exogenous ligand

Using beers law, what is the difference between the estimated concentration that we calculate and the actual concentration?

Actual C = estimated C x Dilution factor

How does DNA cleavage occur during apoptosis?

After initiation of the caspase cascade, active caspase 3 cleaves the DNase inhibitor which allows activated DNase to cleave DNA

Which of the following assays could provide direct information about whether cell death in a given sample has occurred specifically due to apoptosis? Annexin V FITC staining only Trypan Blue staining WST1 assay Propidium iodide staining only

Annexin V FITC staining only

Which stain can provide direct information about whether cell death in a given sample has occurred specifically due to apoptosis?

Annexin V staining

List the antagonists

Atropine, Caffeine, Lidocaine

Your lab group generates a BSA standard curve using the Bradford Assay and finds the slope of the best fit line to be 0.5. Your BSA standard concentrations are in mg/mL. You are then given an unknown BSA sample that you have to dilute 1:20 in order to get a reading within the range of your BSA standards. The diluted unknown sample gives an average A595nm reading of 0.28. What is the approximate actual concentration of your unknown sample in mg/mL? Assume a path length value of '1'. A. 1.12 mg/mL B. 11.2 mg/mL C. 0.56 mg/mL D. 1.8 mg/mL

B

Which of the following about microscope objective lenses is NOT true (choose all that apply)? A. The higher the magnification power, the shorter the working distance B. The lower the magnification power, the shorter the working distance C. The larger the NA value, the smaller the R value. D. The smaller the R value, the lower the magnification power.

B and D

What do we use to track the migration of proteins through a gel?

Bromophenol blue - most proteins are not visible during gel electrophoresis without dye since most do not absorb visible wavelength

What are some of the nuclear changes that occur during apoptosis?

DNA and nuclear fragmentation

Intrinsic apoptosis pathway

DNA damage activates P53 which activates Bax --> cyt. C released from mitochondria --> caspase 9 activated --> caspase 3 activated --> apoptosis

Which of the following is typically caused by an activated caspase 3 enzyme? Choose ALL that apply. DNA fragmentation, DNA repair, Increased cell proliferation, cleavage of the actin cytoskeleton

DNA frag and cleavage of the actin cytoskeleton

Which of the following is typically caused by an activated caspase 3 enzyme? Choose ALL that apply. DNA fragmentation DNA repair cleavage of the actin cytoskeleton increased cell proliferation

DNA fragmentation; cleavage of the actin cytoskeleton

Movement of a charged molecule in an electric field equation (gel electrophoresis)

Ez = fv E = force of the electric field z = net charge on the molecule f = frictional coefficient v = velocity of the migrating particle

True or False. In a phase contrast microscope, the phase plate causes undiffracted light waves to undergo a phase shift by 1/2 wavelength.

False

Which of the following is unique to microplate readers (Choose all that apply)? Incident light passes through the bottom of the sample vessel. . A sample's volume can influence the light path length. Light path length depends on sample vessel width. A specific wavelength of light can be chosen

Incident light passes through the bottom of the sample vessel. . A sample's volume can influence the light path length.

If a given drug is known to be an AGONIST of a heart cell receptor in the sympathetic cell communication pathway, this drug would be expected to:

Increase heart rate

What are the 3 steps of apoptosis

Initiation --> execution --> phagocytosis

Effect of a comp. inhibitor on Km, Vmax, and 1/2Vmax

Km is increased Vmax and 1/2 Vmax are unchanged

What is the relationship between Km and the enzymes affinity for its substrate

Km is inversely proportional to the enzyme affinity

What is the difference between magnification and resolution?

Magnification is the ability to make small objects seem larger, such as making a microscopic organism visible. Resolution is the ability to distinguish two objects from each other.

A dying cell exhibits the following features: swelling of cytoplasm and organelles, rupture of cell membrane that leads to inflammation. This cell is most likely undergoing:

Necrosis

What are some of the differences between necrosis and apoptosis?

Necrosis: - cell swells due to comprimised membrane --> cell lysis --> release of cellular components Apoptosis: - cell shrinkage + chromatin condensation --> cell blebbing --> phagocytosis

List the agonists

Nicotine, Acetylcholine, Epinephrine

In this technique, the amount of colorimetric product produced is directly proportional to the number of living, metabolically active cells in a given sample. Wright Staining Caspase colorimetric assay Annexin V FITC / Propidium Iodide staining None of the above.

None of the above.

Formula to calculate R for objective lenses

R = wavelength / NA - R = the minimal distance that 2 objects can be apart in order for them to resolved as separate (resolution power) - NA = numerical aperture of the lens

Which of the following blood cell/components would originally derive from a myeloid progenitor cell? Choose all that apply. T cells, RBC's, platelets, lymphoblasts

RBC's and platelets

Which of the following blood cell/components would originally derive from a myeloid progenitor cell? Choose all that apply. T cells Red blood cells platelets lymphoblasts

Red blood, cells platelets

In the ___________________ cell communication pathway, a neuron from the central nervous system (CNS) would release the neurotransmitter, ______________, which would bind ___________receptors within autonomic ganglia and cause the release of _________________. Sympathetic ; acetylcholine ; nicotinic; norepinephrine Parasympathetic ; acetylcholine ; nicotinic ; norepinephrine Sympathetic ; norepinephrine ; adrenergic ; acetylcholine None of the above.

Sympathetic ; acetylcholine ; nicotinic; norepinephrine

Which of the following is true about the role of caspase enzymes in apoptosis? Choose all that apply. They act as proteases. Caspase 8 generally acts as an initiator caspase. They all undergo proteolytic cleavage upon activation. Caspase 3 is considered to be the universal caspase initiator.

They act as proteases. Caspase 8 generally acts as an initiator caspase. They all undergo proteolytic cleavage upon activation.

Why is phase contrast microscopy used instead of a regular compound light microscope?

To have enough contrast to see a specimen using a light microscope, we have to use a dye. Doing so kills the sample. - Phase contrast allows us to view the specimen without staining it so we can view it while it is alive - light is moved out of phase to illuminate contrast between things that you are looking at

Which of the following techniques is useful ONLY in determining whether cell death of any kind has occurred? Choose all that apply. Trypan blue staining Annexin V / Propidium Iodide Assay Caspase Colorimetric Assay WST-1 colorimetric assay

Trypan blue staining WST-1 colorimetric assay

In SDS PAGE, the dye, bromophenol blue enables:

Visualization of sample migration during electrophoresis

In SDS PAGE, the dye, bromophenol blue enables: Visualization of individual proteins following electrophoresis Visualization of individual proteins during electrophoresis Visualization of sample migration during electrophoresis Quantification of the amount of protein loaded per well, following electrophoresis

Visualization of sample migration during electrophoresis

Effect of a non-comp. inhibitor on Km, Vmax, and 1/2Vmax

Vmax and 1/2vmax are reduced, Km is unchanged

Which of the assays we used in lab are useful ONLY in determining whether cell death of any kind has occurred? Choose all that apply.

WST-1 colorimetic assay and trypan blue staining

What is critical illumination in phase contrast microscopy?

When the condensor annulus is alligned perfectly with the phase plate

Dielectric constant

a value that describes the polarity of a given substance, such that the higher the polarity of the substance, the higher its constant value is

Which of the following is true about the role of caspase enzymes in apoptosis? Choose all that apply. they act as proteases, caspase 8 generally acts as an initiator caspase, they all undergo proteolytic cleavage upon activation, caspase 3 is considered to be the universal caspase initiator.

all except the last one

What does spectrophotometry measure?

amount of light transmitted or absorbed by a substance in solution

______________ are drugs that can mimic an endogenous ligand but block receptor activation.

antagonists

Cell death by suicide is called what

apoptosis - can occur by internal or external signals

Whats the relationship between light absorbance and concentration?

at specific wavelengths, light absorbance is proportional to concentration (linear relationship)

What is BSA and what are some if its uses in cell bio

bovine serum albumin 1) removing toxic substances 2) used in cell culture protocols 3) can be used as a carrier of small molecules

How do we decrease a protein's solubility in solution?

by promoting hydrophobic aggregation of the proteins 1) denature the protein 2) dehydrate the solvation shell of water molecules around the folded protein

Antagonist drug

can compete for ligand binding to the receptor and block receptor activation

Leukemia

cancer that manifests in immature WBC called blast cells in the bone marrow

Myeloma

cancer that manifests in immature plasma cells within the bone marrow

Lymphoma

cancer that manifests in lympocyte cells

Basics of how gel electrophoresis works

charged molecules migrate in an electric field, permitting the separation of nucleic acids and proteins on the basis of size - distance traveled by these molecules compared to standards can give us an idea of their size

Which of the following forms a part of the structure known as the apoptosome? caspase 3 cytochrome C Bcl2 p53

cytochrome C

Extrinsic apoptosis pathway

death signals bind to death receptors on the plasma membrane --> pro-caspase 8 is activated and becomes caspase 8 --> activates caspase 3 --> apoptosis

As magnification power increases, field of view ____

decreases

By what factor does field of view diameter change as magnification increases from 4x to 10x? from 4x to 40x?

decreases by factors of 2.5 and 10

Primary protein structure

determined by the linear order of its amino acids

Consider the equation, Ez = fv equation, which describes the movement of a molecule through an electric field. Which of the following equation components most directly involves the polyacrylamide gel component in SDS PAGE? E z f v

f

Precipitation

formation of a solid in solution

What is the formula for converting RPM into RCF

g = (1.118x10^-5) x Radius (cm) x RPM^2

Cardiac pacemaker cells are able to facilitate conduction because they (choose all that apply): they are neuronal cells have gap junctions are connected by channels that allow the free passage of ions/small moleucles are capable of selfgenerated contractions

have gap junctions are connected by channels that allow the free passage of ions/small moleucles are capable of selfgenerated contractions

Blood and lymph cells arise from ____ stem cells within the bone marrow

hematopoietic

low Km = _____ substrate affinity of the enzyme

high affinity

Nucleases

hydrolyze nucleic acids

Proteases

hydrolyze proteins

Lipases

hydrolzye lipids

If a given drug is known to be an AGONIST of a heart cell receptor in the sympathetic cell communication pathway, this drug would be expected to: increase heart rate decrease heart rate

increase heart rate

Competitive inhibition

inhibitor binds competitively for the active site but does not result in product formation - presence of this inhib. has NO effect on the enzymes innate catalytic ability. So higher [substrate] can overcome the effects of the inhibitor - reversible

What rose does caspase 3 play in forming apoptotic bodies during apoptosis?

it cleaves proteins of the cytoskeleton causing the cell to lose its structure. Other proteins then cause the cell to collapse into apoptotic bodies

What happens to working distance as magnification power increases?

it decreases

How does spectrophotometry work for protein quantification?

it determines the [ ] of a substance in solution by way of the fact that dissolved substances will absorb light of a specific wavelength, and thus, decrease the amount of light that passes through that sample. - concentration of an absorbing substance can be determined by measuring the intensity of light before and after it passes through solution

3 types of cancers of the blood and immune system

leukemia, lymphoma, and myeloma

What are the main components of a typical specttrophotometer?

light source, means of selecting a single wavelength of light, sample vessle through which light passes, detector that recieves the transmitted light

High Km = _____ substrate affinity of the enzyme

low affinity

The three main types of blood cancer are (choose 3 from the choices below): lymphomas adenomas leukemias myelomas blastomas

lymphomas, leukemias, myelomas

A dying cell exhibits the following features: swelling of cytoplasm and organelles, rupture of cell membrane that leads to inflammation. This cell is most likely undergoing: apoptosis necrosis

necrosis

Cell death by injury is called

necrosis - can happen by mechanical damage or exposure to toxic chemicals

A BSCI330 laboratory class conducts an enzyme kinetics experiment using a given enzyme and increasing amounts of an inhibitor. They generate the two curves below. enzyme kinetics 1.png According this data, what type of inhibitor is being used?

noncompetitive

Interactions among _____ side chains drive protein folding in aqueous environments.

nonpolar (hydrophobic

What does wright stain help us to visualize?

nuclear fragmentation of the cell

Which of the following is NOT true about the caspase colorimetric assay performed during the apoptosis project (week 3)? p-Nitroaniline light absorbance was measured because this compound is a natural product formed inside dying cells as a result of caspase activity. Cell lysates provided the source of caspase enzymes in this assay. The artificial substrates provided in the assay contained p-Nitroaniline, which was only released in the presence of active caspases. This assay could be useful in determining whether an intrinsic or extrinsic apoptosis pathway has been induced in a cell sample.

p-Nitroaniline light absorbance was measured because this compound is a natural product formed inside dying cells as a result of caspase activity.

A student would like to transfer 0.2 mL of blood into a microcentrifuge tube. She grabs a micropipette and adjusts the volume window such that it it reads: 020. This would give her the correct volume if her micropipette is a: p1000 p20 p200

p1000

A sample of blood plasma was mixed with 100% Ethanol, and a precipitate formed. The sample was then spun in a microcentrifuge. Following this spin, the proteins that lost their hydration shells should be in the: supernatant pellet

pellet

Active site

portion of the enzyme where the substrate binding occurs

Trypsin

protease that cleaves a peptide bond after an arginine or lysine is present

Overall protein structure / makeup

proteins are polymers consisting of strings of amino acid monomers covalently linked together via peptide bonds

What does RCF stand for and what does it depend on

relative centrifugal force - describes the amount of accelerative force applied to the sample - force is measured in some value x, the standard acceleration due to gravity - depends on the radius of rotor and the RPM of rotor - measured in g's

What did we use the dot blot assay to test for?

release of cytochrome C

What does RPM stand for and what does it depend on

revolutions per minute - simply describes the rotational speed of rotor - does not depend on rotor size - is only constant for centrifuges using the same rotor size

2 methods we used to dehydrate solvation shells around proteins

salting out and changing the dielectric constant of the solvent

Salting out method

using a salt that effectively competes with proteins for water solvation, causing protein aggregation and precipitation

Changing the dielectric constant method

using an organic solvent that lowers the effectiveness of water solvation by separating water molecules from 1 another, causing protein aggregation and precipitation