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PSYC midterm 2 > Bio lab final > Flashcards

Bio lab final Flashcards

1
Q

What percentage of bleach to dilute biological spills and for how long?

A

10% for 2 minutes

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2
Q

What did we use methylene blue for?

A

Making a standard curve in the first lab

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3
Q

Function of standard curves and how they are made

A

Standard curves are prepared with known concentration of a substance. The equation of the line can be used to interpolate (determine) the unknown concentration of the same substance.

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4
Q

What do standard curves demonstrate?

A

The relationship between known concentrations and absorbance

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5
Q

Does the relationship of a standard curve have to be linear?

A

yes

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6
Q

What is the absorption maximum and when does it have to be determine?

A

The wavelength at which light is maximally absorbed by a specific molecule is called its absorption maximum

Has to be determined before the curve is made

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7
Q

When making standard curves where do the major and minor lines go

A

Major outside
Minor inside

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8
Q

Why do we measure wavelength at the absorption maximum?

A

Ensures highest sensitivity and minimize deviations from Beer’s Law

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9
Q

What are standards?

A

Known concentration of a substance

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10
Q

When are absorption values proportional to the concentration of a substance?

A

the absorption maxima is known and used to measure the absorbance
the appropriate control is used as a blank
The relationship is linear

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11
Q

What do we use chromogens when measuring absorbance? And what do they do?

A

Because cellular components are usually colourless

When they react with a product they turn colour

The absorption can be used to determine concentration of the substance

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12
Q

Is the compound or concentration known in spectrophotometry?

A

Compound

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13
Q

What is spectrophotometry?

A

Technique that measures absorbance of energy (light), of a molecule in solution using a spectrophotometer

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14
Q

What is Beer’s law and equation

A

The absorbance is proportional to the concentration. More concentrated product have higher absorbances

A=kCL

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15
Q

What is the difference vs absorbance and absorption

A

Absorption is the process of absorbing light

Absorbance is the measure of how much light is absorbed

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16
Q

What is the absorption spectrum?

A

graph of the amount of light a substance absorbs. The maximum absorption is the peaks

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17
Q

Is the control used as a blank in making a standard curve? And what does it contain?

A

The control is used as a blank → contains everything except chromagen → use this to set the spectrophotometer to 0

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18
Q

What are two things we need to make sure when making a standard curve?

A

The concentrations of the standards must be chosen so that they fall within the range of sensitivity of the chromogen being used.

Also,the concentration of the unknown must be within the range of the standard curve.

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19
Q

What physical property do spectrophotometers measure?

A

Measures the transmittance of light of a specific wavelength through a sample and gives a reading of absorbance

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20
Q

What colour tips to use with a P10, P20, P200, and P100 pipette

A

P10 -> white
P20 and P200 –> yellow
P1000 –> blue

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21
Q

P1000 pipette volume

A

800 µL

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22
Q

P1000 pipette volume

A

600 µL

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23
Q

P200 Pipette volume

A

40 µL

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24
Q

P200 pipette volume

A

100 µL

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25
Q

P20 pipette volume

A

10 µL

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26
Q

How to determine sigfigs

A

use the number of significant figures equivalent to the level of precision obtainable by the least precise of your measuring devices.

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27
Q

How many sigfigs in 400? How about 400.0?

A

1 and 4

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28
Q

You are given a 5-mL pipette that is graduated in equal divisions of 0.1 mL, and 10 g of sodium crystals. You are asked to add 1 g of salt to 1.0 mL of water, and record the final concentration in g/mL. How should your answer by expressed? (related to sigfigs

A

1g/mL

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29
Q

What is a suspension?

A

A suspension is a mixture of insoluble particles in a suspending fluid, in which the particles will eventually settle out due to gravity if allowed to stand for a sufficient time.

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30
Q

v/v % meaning

A

the number of parts of solute in 100 parts of the solvent or medium

Has to be the same unit

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31
Q

what does v/v 10% aqueous DMSO solution mean

A

10 parts DMSO in 100 parts of water

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32
Q

w/v percentage

A

number of grams of solute in 100 mL of solvent

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33
Q

What does a w/v 10% aqueous sucrose solution mean?

A

10 grams of sucrose were dissolved in a total volume of 100 mL of solvent

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34
Q

What equation to use for dilutions

A

C1V1 =C2V2

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35
Q

You have a stock solution of protein that has a concentration of 1.25 μM, and you need 5.0 mL of a 0.25 μM concentration. How would you make this?

A

Add 1 mL of the 125 uM stock solution to 4 mL of water

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36
Q

What does a 1/100 dilution mean?

A

1 part solute is dissolved in 99 parts water

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37
Q

If you need 240 mL of a 1/10 000 dilution, to determine how much of the original 1/100 solution to use for this, and how much diluent to add?

A

Add 2.4 mL of the original solution and 237.6 mL of diluent

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38
Q

If you were to dissolve 12 g of salt crystals into 200 mL of water, what is the percent (%) concentration of this solution? Is this a weight/volume percentage or a volume/volume percentage?

A

6% and w/v

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39
Q

Four milligrams of a solid chemical are weighed and dissolved in 100 microlitres of water. What percent solution (w/v) was prepared?

A

w/v 4%

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40
Q

A research technician is asked to prepare 120 mL of a 2.5 % aqueous solution of a drug from a stock solution. The stock solution contains 60 g per L of the drug. Show all calculations and units. A) What percent concentration is the stock solution? B) How much of the stock solution and distilled water should be used to achieve the desired dilution?

A

6%, 50 mL of stock and 70 mL of water

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41
Q

What must we use with a 100X microscope?

A

immersion oil

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42
Q

What is Koehler illumination

A

a standardized procedure used to obtain uniform illumination, thus ensuring the
best resolution and image quality possible.

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43
Q

What is a fluorophore/ fluorochrome?

A

molecules that absorb light at a lower wavelength and emit light of a longer wavelength

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44
Q

What does a dichroic mirror do

A

Uses a dichroic mirror to reflect the short wavelengths onto the specimen and transmits the longer wavelengths to the camera

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45
Q

What does DAPI bind to? Where is it seen? what colour does it appear?

A

Binds to A-T rich regions of DNA → shows nucleus in blue

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46
Q

What wavelength is DAPI excited by and what does it emit?

A

Excited by ultraviolet light (358 nm) and emits a blue light (461 nm)

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47
Q

What does Phalloidin-rhodamine bind to? Where is it seen? What colour does it appear?

A

Binds to F-actin, a component of the cytoskeleton

In red

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48
Q

What part of Phalloidin-rhodamine binds to F-actin and what part glows

A

Phalloidin binds to F-actin.

Rhodamine is a family of fluorescent dyes

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49
Q

What did PFA (paraformaldehyde) do in our fluorescent microscopy lab?

A

Fix the cells and permeabilize the cell membranes so the eyes could enter

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50
Q

What wavelength is Phalloidin-rhodamine excited by? What does it omit?

A

excited at 540nm and emits a red light (at 565nm)

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51
Q

Can a compound microscope see membranes?

A

No

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52
Q

Do we use the coarse adjustment knob to focus the 40X lens

A

no

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53
Q

Should we start viewing specimens with the lowest power objective first?

A

Yes

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54
Q

Name the parts (except 2)

A
  1. eyepiece
  2. objective lens
  3. light intensity control
  4. condenser focus knob
  5. fine focus knob
  6. coarse focus knob
  7. stage
  8. condenser iris diaphragm
  9. condenser entering screws
  10. condenser
  11. field iris diaphragm
  12. light source
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55
Q

Field iris diaphragm function?

A

controls the diameter of light field

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56
Q

coarse and fine focus knob

A

adjust the distance between the objective lens and the specimen

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57
Q

Condenser function

A

a series of lenses that focuses the light onto the specimen slide

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58
Q

Condenser focus knob function

A

Moves the condenser up and down at different focal length top get optimal illumination

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59
Q

condenser centring screws function

A

center the condenser so that the source light is centered in the field of view

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60
Q

Condenser iris diaphragm function

A

controls the diameter of the cone of light entering the objective lens

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61
Q

Objective lens and what they do

A

Produce an enlarged but inverted projection of the object.

They are designed to be parfocal and parcentral, meaning that the object remains in focus, and in the center of the field,

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62
Q

ocular lenses/eyepiece

A

They further magnify the object by 10X

If you use a 40X objective, the total magnification is 400X.

One of the ocular lenses houses an ocular micrometer scale for measuring objects through the microscope.

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63
Q

What is depth of field. And is it short or longer for low or high power objective lenses?

A

Depth of an object that is in focus.

Shorter for high power

Longer for low power

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64
Q

If you want to focus on a thick object should you use a high power or low power lease

A

low

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65
Q

What is working distance? Do low or high power lenses have shorter or longer working distances

A

the distance between the specimen and the objective lens.

High power lenses have a shorter working distance and low power have a longer working distance

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66
Q

What is field of view? Do low or high power lenses have larger or smaller fields of view?

A

The diameter of the visible area seen through the microscope. higher power lenses have a smaller filed diameter

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67
Q

Does fluorescent microscopcy measure absorbed or reflected light?

A

Reflected

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68
Q

What channels do we use for DAPI and Phalloidin-Rhodamine stain on the fluorescent microscope?

A

Blue for DAPI and red for Phalloidin-Rhodamine

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69
Q

There are three modes on the fluorescence microscope: live mode, gallery mode, and merge mode. What mode will you use to focus on your cheek cells?

A

Live mode

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70
Q

When focusing on a specimen using the 10X objective I should use the course focus knob first, and then the fine focus knob to bring the object into clear focus?

A

True

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71
Q

What happens during glycolysis

A

glucose is oxidized to two three-carbon molecules of pyruvate

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72
Q

Where does ubiquinone transfer electrons to?

A

Ubiquinone transfers electrons from Complex I and II to Complex III

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73
Q

Where does cytochrome c transfer electrons from

A

Complex III to Complex IV

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74
Q

How can SDH activity be assayed

A

using a ETC inhibitor (azide) and an electron acceptor (DCIP)

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75
Q

What does sodium azide do?

A

Blocks final transfer of electron to oxygen

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76
Q

What is sodium malonate and what does it do?

A

a competitive and reversible inhibitor of SDH

Induces mitochondrial collapse, the release of ROS and free electrons

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77
Q

Maximum wavelength of DCIP

A

600 nm

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78
Q

What is DCIP reduced to

A

DCIPH2

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79
Q

How does DCIP measure enzyme activity?

A

Indirect measure

Electrons that would normally got o oxygen react with DCIP

A standard curve is used to determine DCIP concentration

The rate of change of DCIP concentration is proportional to the rate of SDH activity

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80
Q

What is differential centrifugation used for

A

to separate components of a suspension

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81
Q

Why does differential centrifugation work?

A

Since different cellular components have different size, shape and density, the effect of gravity will be different

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82
Q

at 2000 rpm what is pelleted

A

whole cells, nuclei

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83
Q

at 10000 rpm what is pelleted

A

mitochondria, cell membrane

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84
Q

at 40000 rpm what is pelleted

A

ribosomes

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85
Q

Are separations made by differential centrifugation pure?

A

NO

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86
Q

Why do we keep our isolated cellular organelles on ice?

A

Because they break down via autolysis

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87
Q

What is RCF? Where is it measured from?

A

Relative centrifugal force generated at a given beed for a radius of rotation

Center of the axis of rotation to the middle of centrifugal tube

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88
Q

What is mechanical disruption

A

Fracturing the membrane by grinding cells

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89
Q

Measuring pipette types

A

TD (to deliver) and TC (to contain)

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90
Q

Types of TD pipettes

A

Blowout and gravity

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91
Q

where is the 0 on TD pipettes

A

at the top

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92
Q

what does it mean if a pipettes says 10 mL 1/10

A

it means it has a total measuring capacity of 10 mL and is divided into 1 mL increments

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93
Q

Function of TD blowout pipettes and how do you tell them apart (2 ways)

A

fluid is emptied by “blowing out” the final drop using a pipettor attached to the end

The number closest to the tip is one less than the rated volume of the pipette. the last mL is in the tip

Has a double engraved ring

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94
Q

Function of TD gravity pipette and how to tell it apart

A

No engravings

There will be fluid left in the bottom

the marks go all the way to 10

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95
Q

What are the functions of the Peleus bulb buttons

A

Press A and expel air
Press S to suck up liquid
Push E to empty pipette

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96
Q

Which of the following statements is true?

Succinate dehydrogenase releases electrons into the electron transport chain, and oxidizes succinate to fumarate in the Krebs cycle.

Succinate dehydrogenase releases electrons into the electron transport chain, and reduces succinate to fumarate in the Krebs cycle.

Succinate dehydrogenase releases electrons into the Krebs, and oxidizes succinate to fumarate in the electron transport chain.

Succinate dehydrogenase releases electrons into the Krebs, and reduces succinate to fumarate in the electron transport chain.

A

A

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97
Q

Which of the following is correct?
Sodium azide blocks the transfer of electrons from cytochrome a3 to oxygen, thus inactivating the electron transport chain.

Sodium azide blocks the transfer of electrons through the enzyme succinate dehydrogenase.

Sodium azide requires the chemical malonate to block the transfer of electrons in the electron transport chain.

A

A

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98
Q

What type of inhibitor is sodium malonate?
competitive and reversible

noncompetitive and reversible

competitive and irreversible

noncompetitive and irreversible

A

A

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99
Q

Where is SDH located

A

inner mitochondrial membrane

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100
Q

What happens to DCIP as it accepts electrons from Complex II

A

. DCIP is reduced to DCIPH2

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101
Q

Why do electrons get passed to DCIPH2?

A. DCIP has a higher electronegative potential than the membrane embedded proteins of the inner mitochondrial membrane and has a greater attraction for electrons.

B. Sodium azide blocks downstream components of the ETC such that they cannot participate in the transfer of electrons. Electrons from Complex II are therefore intercepted by DCIP.

C. DCIP is a competitive inhibitor of succinate and binds to the active site of succinate
dehydrogenase, thereby becoming reduced.

A

B

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102
Q

When using a blowout pipette you have to remove all the fluid from the pipette to dispense the intended volume of fluid.

A

true

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103
Q

In the lab this week, when you want to use the Peleus bulb, you need to first release the air from the bulb by squeezing and pressing valve ____ . When you wish to draw up fluid into the blowout pipette press valve ____. When you wish to transfer the fluid out of the pipette you should press valve _____

A

A, S, E

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104
Q

The average Δ absorbance of DCIP @ 600 nm over 20 minutes was determined to be -0.125/minute. If a DCIP standard curve equation of the line is y = 0.055x, then what is the above rate in μM/minute (micromolar per minute)

A

x= -2.27 uM/minute

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105
Q

In step 8 of the procedure, after collecting ~5 mL of the Mitochondrial-free fraction (MFF), you are asked to discard the remaining supernatant. How did Sarah do this in the video?
A. Sarah poured the supernatant out of the centrifuge tube
B. Sarah used a Pasteur pipette to remove the supernatant
C. Sarah used a blowout pipette to remove the supernatant

A

A

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106
Q

. How is succinate dehydrogenase measured in the lab?

A. Spectrophotometrically and indirectly using the artificial electron acceptor DCIP

B. Spectrophotometrically and directly using the artificial electron acceptor DCIP

C. Spectrophotometrically and directly using FADH

A

A

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107
Q

Why do we refer to the measurement in this activity as indirect?

A

The measurement of enzyme activity is indirect because we are measuring the rate of SDH activity from the rate of decrease of DCIP concentration, which is measured by the rate of change of absorbance as the colour changes from blue to colourless.S

Succinate reacts with FAD to create Fumerate and FADH2. The FADH2 then reacts with DCIP to decrease the colour. This is indirect because the product of the reaction, FADH2 reacts with DCIP of the next reaction. DCIP is not a product of SDH

Since DCIP is not a product of SDH, it I indirect.

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108
Q

What colour does DCIP change from

A

Blue to colourless

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109
Q

Explain why the change in DCIP concentration in the MF-M is the greatest.

A

Maonate induces mitochondrial potential collapse and the production of reactive oxygen species. The free electrons can react with DCIP to turn it into DCIP2.

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110
Q

Non measuring pipettes name

A

pasteur pipette

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111
Q

How large are cell cultures

A

typically in the range of millions or billions of cells per milliliter,

112
Q

Can cells be frozen?

A

yes

113
Q

Can we count a pure cell culture in a hemocytometer?

A

No, we must dilute it first so it is in the countable range

114
Q

What is the countable range for cells in a hemocytometer?

A

1000 cells/mL

115
Q

What are some examples of cell culturing conditions?

A

Temperature
Agitation
Nutrients
Doubling time
Contamination
Waste, die off

116
Q

What temperate are human cells and coli cultured at?

A

37 degrees

117
Q

What temperate is Yeast cultured at

A

30 degrees

118
Q

What is the doubling time of yeast

A

1.8 hours

119
Q

When is something truly sterile?

A

When it does not contain any living things

120
Q

What is the scientific name of yeast?

A

S. cerevisiae

121
Q

What is used to make things sterile (aseptic)

A

autoclave

122
Q

Why do we need to sterilize even if we think our culture is safe (3 reasons)

A

Could have introduced a harmful microbe

Harmless microbes could mutate and become harmful

Some people may be more sensitive to harmful microbes

123
Q

Should we minimize air current in a sterile environment

A

yes since air is not sterile

124
Q

Why do we put opening of containers over a flame?

A

To sterilize and to push air away from the culture

125
Q

Should you place sterile lids on a table

A

no

126
Q

What do we use to determine cell culture concentration and what do we use this for?

A

a hemocytometer to determine cell concentration so we can dilute for plating

127
Q

What do we need to do before we load a hemocytometer

A

dilute

128
Q

W

A
128
Q

What is a serial dilution

A

series of successive dilutions

129
Q

What do you need to do first before you plate cells? (related to serial dilution)

A

In order to prepare a serial dilution for plating cells, you must first determine the concentration of the stock culture (in cells/mL) and then decide what concentration you want to dilute your stock by

From this, you need to determine what your dilution factor should be

130
Q

Do you start with the largest or smallest dilution first and give an example

A

Start with largest

1/2.5 dilution first and the 1/100 dilution second

131
Q

Equation for total dilution factor when stock concentration too required concentration

A

Total dilution factor = (stock concentration in cells/mL) / (Required concentration in cells/ mL)

132
Q

If your stock concentration was determined to be 2.0 X 10^8 cells/mL and you wanted to dilute the stock to 200 cells/mL, then determine the dilution factor

A

(2 X 10^8 cells/mL)/(200 cells/mL) = total dilution factor = 10^6
Written as total dilution 1/1000000

133
Q

Function of hemocytometer

A

A hemocytometer provides a chamber of known area and depth, which allows for cell counting and calculation of the original stock concentration (cells/mL)

134
Q

How much do we usually add to a hemocytometer?

A

10 uL to each side but needs to be diluted prior

135
Q

Do you need to take into account your initial dilution when determining original cell concentration using a hemocytometer?

A

yes

136
Q

What lens do we use to focus on a grid. Then which one to zoom into the squares

A

4X then 40X

137
Q

How many cells should there be on the 5 squares for s statistically accurate measurement? If there isn’t what do you do?

A

50 -150 total (50-200 is fine)

(10-30) each square

dilute again

138
Q

Is the volume in a hemocytometer always the same?

A

yes

139
Q

3 rules for counting cells

A

only daughter cells greater than 50% of the size of the mother cell are counted

Cells that are adherent to each other are counted as being separate cells

If they pass the middle border they don’t get counted (0n it is fine)

140
Q

What is sample error

A

an individual sample can vary randomly from the whole population
due to sample error

ie: not vortexing

141
Q

What squares do we count in the hemocytometer

A

all 5

center and corners

142
Q

What is the area of the 5 squares in the hemocytometer in mm^3

A

0.004x5 = 0.02 mm^3

143
Q

Equation for determining original stock concentration from counted squares in hemocytometer

A

(# of cells in 5 squares)(DF)/ (volume in one square)(# of squares) X (Conversion factor of mm3 to mL)

144
Q

Conversion factor of mm3 to mL

A

1000

145
Q

How many cells per mL do we want to plate

A

1000 cells per mL

146
Q

How much do we plate (volume) and number of cells

A

0.1 mL –> 100 cells

147
Q

How would you calculate the total dilution factor from the stock concentration to 1000 cells/mL

A

Total D.F. = stock concentration (cells/mL)/required concentration (cells/mL)

148
Q

What do you do once you determine that DF to dilute your cells to 1000 cells/mL?

A

Serially dilute the culture

149
Q

What is the maximum volume in each tube for a serial dilution. What is the minimum amount transferred each time?

A

1 mL

10 uL

150
Q

What do the number of colonies represent?

A

The number of cells plated

151
Q

What can the number of colonies be used to back-calculate?

A

the concentration of the stock

152
Q

How to determine plating efficiency?

A

Plating efficiency = number of colonies counted number of cells plated

153
Q

How to determine number of cells plated?

A

take concentration plated and multiply by volume: (1000 cells/mL) (0.1mL) = 100 cells/mL

154
Q

If we counted 38 colonies and we plated 100 cells what is the plating efficiency?

A

38%

155
Q

Equation to determine cell concentration of the stock solution fro number of colonies counted

A

(# of colonies counted / (plating volume) (1/plating efficiency in decimal) (DF)= cells/ mL of stock solution

156
Q

Should your stock concentration determined by the hemocytometer be the same a what you determined by plating?

A

yes

157
Q

If there are some causes that may have effected the amount of colonies that grew

A

Letting cells settle before taking plating volume
Overheating/killing cells while trying to create a sterile environment
Over usage may have damaged the cells Mutations in yeast colony could impact cell viability and replication ability

158
Q

Where to put leftover Yeast culture

A

into yellow biohazard container

159
Q

What % ethanol to clean with

A

70%

160
Q

How many Yeast cells

A

5

161
Q

Air is considered _____(contaminated or sterile), as are test tube lids placed on the countertop.

A

contaminated

162
Q

If you needed to dilute a stock cell culture 1/67, how much of the stock would you add to the tube if it had 660 μL of diluent?

A

10 uL

163
Q

How many cells don’t count here

A

2 and the baby

164
Q

A researcher dilutes a stock culture of cells 1/4000, and adds a 25 μL sample to a hemocytometer. From the
cell count, she determines the concentration of the stock culture to be 10 X 10^9 cells/mL. What was the
total number of cells she counted in 5 squares?

A

50 cells

165
Q

What’s the optimal temperature for S. cerevisiae culture?

A

30 °C

166
Q

. On a hemocytometer, what is the total volume of the fluid
beneath five squares (0.2mm x 0.2mm, 0.1 deep), in cubic
millimeters (mm3)?

A

0.02 mm3

167
Q

A 100 mL yeast culture has a concentration of 4 x107 cells/mL.
The culture is diluted to 2000 cells/mL. 100 μL of diluted culture
is plated and 100 colonies grow on plate. What’s the plating efficiency?

A

50%

168
Q

If you perform a 1 in 10 dilution, what’s the dilution and
dilution factor?

A

1/10 and 10

169
Q

What did we do in lab 6 with RBCs?

A

Fractionate red blood cells and determine protein concentration

170
Q

What are the main methods for determining protein concentration?

A

Last week: Through a series of differential centrifugation steps the plasma, lysate and membrane fractions were collected

The plasma and lysate fractions are serially diluted (to fall within range of the protein standard curve)

The protein concentration of each fraction is determined using spectroscopy and BSA standard curve (known as Bradford Assay)

Adjusted protein concentrations for each fraction are calculated

The plasma fraction is prepared for SDS-PAGE (lab 7)

171
Q

What protein standard is used to determine protein concentration?

A

Known concentrations of bovine serum albumin (BSA)

172
Q

Since we can’t measure BSA using spectrophotometry, what do we do?

A

the protein samples are mixed with a colorimetric substance –> the Bradford Reagent

173
Q

What do we use to make s standard curve to determine protein concentration

A

A protein standard containing a known concentration of protein is mixed with the Bradford Reagent and the absorbance value read

174
Q

What does the Bradford reagent contain?

A

Coomassie Brilliant Blue

175
Q

What does the Bradford reagent bind to quantitively?

A

to arginine (basic) and aromatic amino acids such as tyrosine, phenylalanine and tryptophan

176
Q

What wavelengths does the Bradford reagent shift from when bound

A

470 to 595

177
Q

What is the max wavelength of Bradford reagent?

A

595 nm

178
Q

What do we plot on the BSA standard curve?at what absorbance

A

absorbance at 595 nm and concentration of BSA

179
Q

What will we use the BSA standard curve to calculate?

A

Use the equation of the line to calculate the protein concentration of the plasma, lysate and membrane fractions that you will isolate today from horse red blood cells.

180
Q

What is the BSA standard curve also known as?

A

Brandford assay

181
Q

How do we isolate the plasma, lysate and membrane fraction?

A

differenital centrifugation

182
Q

How do we plot the BSA standard curve?

A

known concentrations of bovine serum albumin to use as a protein standard.

183
Q

What is the extracellular matrix of blood?

A

Plasma

184
Q

What do RBC do in a isotonic saline?

A

In isotonic saline have a net zero exchange of water across the membrane.

185
Q

What do RBC do in a hypotonic solution

A

have a net influx of water causing the cells to lyse

186
Q

What is fractionation?

A

procedure by which cells or tissues are broken down, and their components separated using centrifugation

187
Q

What techniques do we use for blood fractionation (2)

A

osmotic lysis and differential centrifugation

188
Q

What do we separate our blood into

A

Plasma, lysate (RBC cytoplasm), and cell membrane

189
Q

What is separated from the blood at low speeds?

A

Plasma from erythrocytes

190
Q

What is between the plasma and RBC?

A

Leukocytes and platelets (Buffy coat)

191
Q

Once erythrocytes are separated from the plasma, what do we do to them?

A

lysed in a hypotonic solution

192
Q

What do we separate out after lysing the RBC using centrifugation?

A

Lysate and membrane

193
Q

What causes proteins to easily degrade and what do we do to stop this

A

ubiquitous cellular proteases released from cellular compartments following osmotic lysis degrade proteins.

Slowed down by keeping samples cold

194
Q

Is coomassie blue acidic or basic?

A

acidic

195
Q

What do coomassie blue in the Bradford assay bind to

A

binds quantitatively to arginine (basic) and to aromatic amino acids such as tyrosine, phenylalanine and tryptophan residues in the proteins.

196
Q

How long is the Bradford assay reliable for

A

about an hour

197
Q

What protein standard do we use to make our standard curve in the Bradford assay?

A

bovine serum albumin (BSA), which is extracted from the blood of cattle

198
Q

Why do we use BSA specifically. Could something else be used?

A

Used because it contains lots of arginine and aromatic amino acids

199
Q

What is the Bradford assay?

A

A method used for determining protein concentration using coomassie blue and a protein standard, such as BSA, with lots of arginine and aromatic AA.

200
Q

What happens when the Bradford reagent (coomassie blue) binds to BSA or proteins

A

shifts from brown to blue

201
Q

What did we use the Bradford assay for?

A

To determine the protein concentration in our membrane, lysate, and plasma samples

202
Q

What proteins doe the plasma consist of

A

serum albumin, globulins, clotting and hormone-binding proteins

203
Q

What proteins are found in the lysate?

A

hemoglobin

204
Q

What proteins are found in the membrane

A

membrane proteins and cytoskeleton proteins

205
Q

What do we do with the interpolated protein concentrations we measure using the equation of the line during the Bradford assay?

A

Multiply it by the dilution factor to get the adjusted protein concentration

206
Q

Why do we keep centrifuging the RBC pellet wit isotonic saline?

A

Removes plasma proteins from the RBC

207
Q
A
208
Q

After we lyse the cells using hypotonic saline and centrifuge, what is in the supernatant and what is the pellet

A

lysate with hemoglobin and membrane is the pellet

209
Q

What do we continue to wash the membrane fraction with hypotonic saline. What colour should it be when we are done?

A

To remove lysate proteins so they don’t affect the accuracy of the protein concentration

Light pink or clear

210
Q

When making their BSA standard curve, do we add Bradford reagent to every sample? Do we add BSA to every sample?

A

Yes

No, the concentrations of BSA varies

211
Q

What absorbance do we use for the BSA standard curve?

A

592 nm

212
Q

Do we use the concentration of Bradford reagent when calculating final BSA concentration? Why?

A

No, because each tube contains the same amount of Bradford reagent and we are interested in knowing the concentration of BSA.

213
Q

What do we put in the BSA standard curve?

A

BSA protein concentration and absorbance

214
Q

What do we have to do to our protein fraction prior to determining protein concentration? Why?

A

We must perform a serial dilution to the plasma and lysate because the protein concentration is too high for the standard curve.

215
Q

What do we dilute the plasma, lysate, and membrane fraction to? What what dilution steps did we use?

A

Plasma 1/100
Lysate 1/1000
Membrane 1/1

1/10 with a total volume of 100 uL

216
Q

What do we do with the membrane, plasma and lysate fractions after we dilute them? What do we use as our blank?

A

100 uL of hypotonic saline
Add 1000 uL of Bradford to all of our samples and let sit for 5 minutes

217
Q

How long do we let the proteins with the Bradford reagent sit for before reading absorbance?

A

5 minutes

218
Q

What do we do with the absorbance values we measured for the plasma, membrane and lysate fractions to get them to the true concentration (adjusted concentration)

A

input the absorbances into the equation of the line and solve for x. Then multiply to the dilution factor

219
Q

Describe a suitable control to test the specificity of the Bradford assay for certain amino acids.

A

We would add the Bradford reagent to a sample or protein that does not contain a lot of positively charged amino acids then compare the colour change to a protein known to include lots of positively charged amino acids.

220
Q

What did we do to prepare our plasma sample for SDS page? and dilute it to what?

A

Mix it with 2X sample buffer
1/2 dilution

221
Q

A hypotonic solution results in the movement of water ____
A hypertonic solution results in the movement of water_____
An isotonic solution results in the movement of water ____

A

Into the cell
equally across the cell
out of the cell

222
Q

Protein quantification occurs when Coomassie Brilliant blue binds to any one of the known amino acids.

A

False

223
Q

Observe how the mixture becomes clear after the addition of the hypotonic saline. Why does the sample become more clear?

A

a. Cells are osmotically lysed

224
Q

In planning for a 1/10 dilution, you determine that ____ μL of the fraction should be added to _____μL of hypotonic saline. To achieve the desired total dilution for the plasma and lysate fractions you will have to do a serial dilution using only 1/10 dilution steps. You determine that ____number of 1/10 dilutions would be required to serially dilute the plasma fraction by a factor of 100. You determine that ____number of 1/10 dilutions would be required to serially dilute the lysate fraction by a factor of 1000

A

10 uL, 90 uL, 2, 3

225
Q

What were our protein samples mix with for SDS page?

A

2X sample buffer

226
Q

What does the 2X sample buffer contain?

A

Beta-mercaptoethanol:
Sodium dodecyl sulfate (SDS)
Bromophenol blue
Glycerol:

227
Q

What is the function of Beta-mercaptoethanol in 2X sample buffer

A

denatures the protein by cleaving disulphide bonds

228
Q

What is the function of SDS in the 2X sample buffer?

A

denatures and binds to proteins giving them a negative charge

229
Q

What is the function of glycerol in 2x sample buffer?

A

adds density to the sample

230
Q

What is the function of Bromophenol blue in the 2x sample buffer?

A

tacking dye that allows us to see the sample moving through the gel

231
Q

What is the function of the protein standard/ladder? And what does it contain? is it mixed with 2x sample buffer?

A

is used to estimate the approx. weights of the protein samples.

contains a mix of proteins of KNOWN molecular weight (kDa).

YEs

232
Q

What do we use coomassie brilliant blue for after electrophoresis? and what does it bind to?

A

Coomassie brilliant blue dye reacts and binds to arginine and
aromatic amino acids

We stain the gel with it to reveal the proteins

233
Q

What are the two layers of an SDS-PAGE gel

A

Stacking and resolving gel

234
Q

how will we the results of SDS-PAGE to determine?

A

The protein bands identified following Coomassie blue staining will be compared to a molecular weight ladder of proteins of known sizes to determine the size of the unidentified purified proteins

235
Q

What is electrophoresis?

A

a method commonly used to separate charged molecules such as proteins

236
Q

Where will negatively charged molecules migrate during electrophoresis (no SDS)? What about positively charged?

A

negatively charged molecules will migrate towards the positive pole (anode) –> down

Positively charged molecules will migrate towards the negative pole (cathode)

237
Q

Why are different bands formed in SDS-PAGE gel?

A

olecules that differ in charge and size will migrate to form distinct bands in a gel matrix

238
Q

What does SDS-PAGE stand for?

A

sodium dodecyl sulfate polyacrylamide gel electrophoresis

239
Q

What is SDS-PAGE used for

A

SDS PAGE is used to discover the molecular weights of proteins in a protein sample

240
Q

What is the gel made out of for SDS page?

A

polyacrylamide gel matrix

241
Q

What does SDS do to the proteins?

A

denatures and binds to proteins leading to an overall negative charge (-1 charge)

242
Q

How much SDS binds to 1 g of protein and what do this do?

A

It is known that 1.4g of SDS will bind 1g of protein thus SDS-protein complexes will migrate as though they have the same charge-to-mass ratio

243
Q

What is the negative charged during SDS-Page determined by?

A

by SDS and not by the charge of the protein

244
Q

Why is it important that the charge-to-mass ratio is the same for SDS-PAGE

A

When the charge-to-mass ratio is the same, proteins will seperate based on on molecular weight.

245
Q

What is the function of a protein ladder?

A

Ptein standard/ ladder:
A set of protein standards, or proteins of known molecular weights, are run alongside the samples to determine the molecular weights of the protein samples.

246
Q

How do we make the standard curve of the protein standards?

A

We used the protein ladder and use the log of the molecular weights and the distance traveled in the gel.

247
Q

What is the x and y value for the standard curve to determine protein molecular weights?

A

x= molecular weight
y = length

248
Q

What do we do once the gel is completely stained with coomassie blue?

A

de-stain it using water

249
Q

Do small or large proteins move the fastest through the gel?

A

Small

250
Q

What is the function of the stacking layer?

A

To get all of the protein samples lined up so they can enter the resolving layer at exactly the same time

251
Q

What is the function of the running buffer in the tetra cell?

A

allows for conductivity

252
Q

What protein samples did we test in SDS page?

A

membrane,
Plasma
Protein A or B

253
Q

Does the resolving layer contain more acrylamide?

A

yes

254
Q

What does the TMED and APS do when you add it to the gel layers

A

polymerizes them

255
Q

What way does the small plate face when making a SDS gel

A

you

256
Q

What is our standard curve of protein weights based on?

A

tandard curve of the log10 of molecular weights of the protein standard against the distance traveled from the top of the resolving layer.

257
Q

What do we do with our protein standard curve for molecular weight?

A

Input the length of one of our sample for y and find X

258
Q

Once you find the value X, the molecular weight, what do you have to do

A

10^x

259
Q

What do do if spills are noxious or irrittating?

A

evacuate

260
Q

What to do with a spill of microorganisms

A

dilute the spill with a 10% solution of bleach

261
Q

If there is a minor fire what do you do

A

call for help, attempt to control it, then if it isn’t controlled in 1 minute leave

262
Q

What are type A extinguishers used for

A

ONLY for trash, wood or paper combustibles.

263
Q

What are type B extinguishers used for?

A

ONLY grease or liquids

264
Q

What are type C fire extinguishers used for

A

ONLY electrical fires

265
Q

What are type ABC extinguishers used for

A

all types

266
Q

What are type BC fire extinguishers used for?

A

electrical, oil, grease, liquid

267
Q

What is WHMIS?

A

Workplace Hazardous Materials Information System

268
Q

What are the four components of WHMIS

A

Product Labeling
Labware labeling
Material Safety Data Sheets (MSDSs)
WHMIS Education and Training Programs

269
Q

WHAT IS THIS?

A

Compressed gas

270
Q

What is this

A

flammable or combustible materials?

271
Q

What is this? and what does it do

A

oxidizing materials

Increases risk of fire if they come in contact with flammable or combustion able materials

272
Q

What is this?

A

materials causing serious toxic effects. may cause death in small amounts

273
Q

What is this? What does it mean

A

Materials causing other toxic effects?

can cause long term health effects

asbestos

274
Q

What is this

A

Biohazardous infectious materials

275
Q

What is this

A

Corrosive materials. Products which may self-react upon standing.

276
Q

Where will a positively charged protein migrate in SDS page

A

A positively charged protein in SDS page will become negative due to the SDS and will migrate to the anode.

PSYC midterm 2 (6 decks)

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