Bio lab final Flashcards
1
Q
What percentage of bleach to dilute biological spills and for how long?
A
10% for 2 minutes
2
Q
What did we use methylene blue for?
A
Making a standard curve in the first lab
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.
4
Q
What do standard curves demonstrate?
A
The relationship between known concentrations and absorbance
5
Q
Does the relationship of a standard curve have to be linear?
A
yes
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
7
Q
When making standard curves where do the major and minor lines go
A
Major outside
Minor inside
8
Q
Why do we measure wavelength at the absorption maximum?
A
Ensures highest sensitivity and minimize deviations from Beer’s Law
9
Q
What are standards?
A
Known concentration of a substance
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
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
12
Q
Is the compound or concentration known in spectrophotometry?
A
Compound
13
Q
What is spectrophotometry?
A
Technique that measures absorbance of energy (light), of a molecule in solution using a spectrophotometer
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
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
16
Q
What is the absorption spectrum?
A
graph of the amount of light a substance absorbs. The maximum absorption is the peaks
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
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.
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
20
Q
What colour tips to use with a P10, P20, P200, and P100 pipette
A
P10 -> white
P20 and P200 –> yellow
P1000 –> blue
21
Q
P1000 pipette volume
A
800 µL
22
Q
P1000 pipette volume
A
600 µL
23
Q
P200 Pipette volume
A
40 µL
24
Q
P200 pipette volume
A
100 µL
25
P20 pipette volume
10 µL
26
How to determine sigfigs
use the number of significant figures equivalent to the level of precision obtainable by the least precise of your measuring devices.
27
How many sigfigs in 400? How about 400.0?
1 and 4
28
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
1g/mL
29
What is a suspension?
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.
30
v/v % meaning
the number of parts of solute in 100 parts of the solvent or medium
Has to be the same unit
31
what does v/v 10% aqueous DMSO solution mean
10 parts DMSO in 100 parts of water
32
w/v percentage
number of grams of solute in 100 mL of solvent
33
What does a w/v 10% aqueous sucrose solution mean?
10 grams of sucrose were dissolved in a total volume of 100 mL of solvent
34
What equation to use for dilutions
C1V1 =C2V2
35
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?
Add 1 mL of the 125 uM stock solution to 4 mL of water
36
What does a 1/100 dilution mean?
1 part solute is dissolved in 99 parts water
37
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?
Add 2.4 mL of the original solution and 237.6 mL of diluent
38
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?
6% and w/v
39
Four milligrams of a solid chemical are weighed and dissolved in 100 microlitres of water. What percent solution (w/v) was prepared?
w/v 4%
40
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?
6%, 50 mL of stock and 70 mL of water
41
What must we use with a 100X microscope?
immersion oil
42
What is Koehler illumination
a standardized procedure used to obtain uniform illumination, thus ensuring the
best resolution and image quality possible.
43
What is a fluorophore/ fluorochrome?
molecules that absorb light at a lower wavelength and emit light of a longer wavelength
44
What does a dichroic mirror do
Uses a dichroic mirror to reflect the short wavelengths onto the specimen and transmits the longer wavelengths to the camera
45
What does DAPI bind to? Where is it seen? what colour does it appear?
Binds to A-T rich regions of DNA → shows nucleus in blue
46
What wavelength is DAPI excited by and what does it emit?
Excited by ultraviolet light (358 nm) and emits a blue light (461 nm)
47
What does Phalloidin-rhodamine bind to? Where is it seen? What colour does it appear?
Binds to F-actin, a component of the cytoskeleton
In red
48
What part of Phalloidin-rhodamine binds to F-actin and what part glows
Phalloidin binds to F-actin.
Rhodamine is a family of fluorescent dyes
49
What did PFA (paraformaldehyde) do in our fluorescent microscopy lab?
Fix the cells and permeabilize the cell membranes so the eyes could enter
50
What wavelength is Phalloidin-rhodamine excited by? What does it omit?
excited at 540nm and emits a red light (at 565nm)
51
Can a compound microscope see membranes?
No
52
Do we use the coarse adjustment knob to focus the 40X lens
no
53
Should we start viewing specimens with the lowest power objective first?
Yes
54
Name the parts (except 2)
1. eyepiece
3. objective lens
4. light intensity control
5. condenser focus knob
6. fine focus knob
7. coarse focus knob
8. stage
9. condenser iris diaphragm
10. condenser entering screws
11. condenser
12. field iris diaphragm
13. light source
55
Field iris diaphragm function?
controls the diameter of light field
56
coarse and fine focus knob
adjust the distance between the objective lens and the specimen
57
Condenser function
a series of lenses that focuses the light onto the specimen slide
58
Condenser focus knob function
Moves the condenser up and down at different focal length top get optimal illumination
59
condenser centring screws function
center the condenser so that the source light is centered in the field of view
60
Condenser iris diaphragm function
controls the diameter of the cone of light entering the objective lens
61
Objective lens and what they do
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,
62
ocular lenses/eyepiece
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.
63
What is depth of field. And is it short or longer for low or high power objective lenses?
Depth of an object that is in focus.
Shorter for high power
Longer for low power
64
If you want to focus on a thick object should you use a high power or low power lease
low
65
What is working distance? Do low or high power lenses have shorter or longer working distances
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
66
What is field of view? Do low or high power lenses have larger or smaller fields of view?
The diameter of the visible area seen through the microscope. higher power lenses have a smaller filed diameter
67
Does fluorescent microscopcy measure absorbed or reflected light?
Reflected
68
What channels do we use for DAPI and Phalloidin-Rhodamine stain on the fluorescent microscope?
Blue for DAPI and red for Phalloidin-Rhodamine
69
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?
Live mode
70
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?
True
71
What happens during glycolysis
glucose is oxidized to two three-carbon molecules of pyruvate
72
Where does ubiquinone transfer electrons to?
Ubiquinone transfers electrons from Complex I and II to Complex III
73
Where does cytochrome c transfer electrons from
Complex III to Complex IV
74
How can SDH activity be assayed
using a ETC inhibitor (azide) and an electron acceptor (DCIP)
75
What does sodium azide do?
Blocks final transfer of electron to oxygen
76
What is sodium malonate and what does it do?
a competitive and reversible inhibitor of SDH
Induces mitochondrial collapse, the release of ROS and free electrons
77
Maximum wavelength of DCIP
600 nm
78
What is DCIP reduced to
DCIPH2
79
How does DCIP measure enzyme activity?
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
80
What is differential centrifugation used for
to separate components of a suspension
81
Why does differential centrifugation work?
Since different cellular components have different size, shape and density, the effect of gravity will be different
82
at 2000 rpm what is pelleted
whole cells, nuclei
83
at 10000 rpm what is pelleted
mitochondria, cell membrane
84
at 40000 rpm what is pelleted
ribosomes
85
Are separations made by differential centrifugation pure?
NO
86
Why do we keep our isolated cellular organelles on ice?
Because they break down via autolysis
87
What is RCF? Where is it measured from?
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
88
What is mechanical disruption
Fracturing the membrane by grinding cells
89
Measuring pipette types
TD (to deliver) and TC (to contain)
90
Types of TD pipettes
Blowout and gravity
91
where is the 0 on TD pipettes
at the top
92
what does it mean if a pipettes says 10 mL 1/10
it means it has a total measuring capacity of 10 mL and is divided into 1 mL increments
93
Function of TD blowout pipettes and how do you tell them apart (2 ways)
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
94
Function of TD gravity pipette and how to tell it apart
No engravings
There will be fluid left in the bottom
the marks go all the way to 10
95
What are the functions of the Peleus bulb buttons
Press A and expel air
Press S to suck up liquid
Push E to empty pipette
96
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
97
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
98
What type of inhibitor is sodium malonate?
competitive and reversible
noncompetitive and reversible
competitive and irreversible
noncompetitive and irreversible
A
99
Where is SDH located
inner mitochondrial membrane
100
What happens to DCIP as it accepts electrons from Complex II
. DCIP is reduced to DCIPH2
101
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.
B
102
When using a blowout pipette you have to remove all the fluid from the pipette to dispense the intended volume of fluid.
true
103
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, S, E
104
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)
x= -2.27 uM/minute
105
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
106
. 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
107
Why do we refer to the measurement in this activity as indirect?
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.
108
What colour does DCIP change from
Blue to colourless
109
Explain why the change in DCIP concentration in the MF-M is the greatest.
Maonate induces mitochondrial potential collapse and the production of reactive oxygen species. The free electrons can react with DCIP to turn it into DCIP2.
110
Non measuring pipettes name
pasteur pipette
111
How large are cell cultures
typically in the range of millions or billions of cells per milliliter,
112
Can cells be frozen?
yes
113
Can we count a pure cell culture in a hemocytometer?
No, we must dilute it first so it is in the countable range
114
What is the countable range for cells in a hemocytometer?
1000 cells/mL
115
What are some examples of cell culturing conditions?
Temperature
Agitation
Nutrients
Doubling time
Contamination
Waste, die off
116
What temperate are human cells and coli cultured at?
37 degrees
117
What temperate is Yeast cultured at
30 degrees
118
What is the doubling time of yeast
1.8 hours
119
When is something truly sterile?
When it does not contain any living things
120
What is the scientific name of yeast?
S. cerevisiae
121
What is used to make things sterile (aseptic)
autoclave
122
Why do we need to sterilize even if we think our culture is safe (3 reasons)
Could have introduced a harmful microbe
Harmless microbes could mutate and become harmful
Some people may be more sensitive to harmful microbes
123
Should we minimize air current in a sterile environment
yes since air is not sterile
124
Why do we put opening of containers over a flame?
To sterilize and to push air away from the culture
125
Should you place sterile lids on a table
no
126
What do we use to determine cell culture concentration and what do we use this for?
a hemocytometer to determine cell concentration so we can dilute for plating
127
What do we need to do before we load a hemocytometer
dilute
128
W
128
What is a serial dilution
series of successive dilutions
129
What do you need to do first before you plate cells? (related to serial dilution)
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
Do you start with the largest or smallest dilution first and give an example
Start with largest
1/2.5 dilution first and the 1/100 dilution second
131
Equation for total dilution factor when stock concentration too required concentration
Total dilution factor = (stock concentration in cells/mL) / (Required concentration in cells/ mL)
132
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
(2 X 10^8 cells/mL)/(200 cells/mL) = total dilution factor = 10^6
Written as total dilution 1/1000000
133
Function of hemocytometer
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
How much do we usually add to a hemocytometer?
10 uL to each side but needs to be diluted prior
135
Do you need to take into account your initial dilution when determining original cell concentration using a hemocytometer?
yes
136
What lens do we use to focus on a grid. Then which one to zoom into the squares
4X then 40X
137
How many cells should there be on the 5 squares for s statistically accurate measurement? If there isn't what do you do?
50 -150 total (50-200 is fine)
(10-30) each square
dilute again
138
Is the volume in a hemocytometer always the same?
yes
139
3 rules for counting cells
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
What is sample error
an individual sample can vary randomly from the whole population
due to sample error
ie: not vortexing
141
What squares do we count in the hemocytometer
all 5
center and corners
142
What is the area of the 5 squares in the hemocytometer in mm^3
0.004x5 = 0.02 mm^3
143
Equation for determining original stock concentration from counted squares in hemocytometer
(# of cells in 5 squares)(DF)/ (volume in one square)(# of squares) X (Conversion factor of mm3 to mL)
144
Conversion factor of mm3 to mL
1000
145
How many cells per mL do we want to plate
1000 cells per mL
146
How much do we plate (volume) and number of cells
0.1 mL --> 100 cells
147
How would you calculate the total dilution factor from the stock concentration to 1000 cells/mL
Total D.F. = stock concentration (cells/mL)/required concentration (cells/mL)
148
What do you do once you determine that DF to dilute your cells to 1000 cells/mL?
Serially dilute the culture
149
What is the maximum volume in each tube for a serial dilution. What is the minimum amount transferred each time?
1 mL
10 uL
150
What do the number of colonies represent?
The number of cells plated
151
What can the number of colonies be used to back-calculate?
the concentration of the stock
152
How to determine plating efficiency?
Plating efficiency = number of colonies counted number of cells plated
153
How to determine number of cells plated?
take concentration plated and multiply by volume: (1000 cells/mL) (0.1mL) = 100 cells/mL
154
If we counted 38 colonies and we plated 100 cells what is the plating efficiency?
38%
155
Equation to determine cell concentration of the stock solution fro number of colonies counted
(# of colonies counted / (plating volume) (1/plating efficiency in decimal) (DF)= cells/ mL of stock solution
156
Should your stock concentration determined by the hemocytometer be the same a what you determined by plating?
yes
157
If there are some causes that may have effected the amount of colonies that grew
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
Where to put leftover Yeast culture
into yellow biohazard container
159
What % ethanol to clean with
70%
160
How many Yeast cells
5
161
Air is considered _____(contaminated or sterile), as are test tube lids placed on the countertop.
contaminated
162
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?
10 uL
163
How many cells don't count here
2 and the baby
164
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?
50 cells
165
What’s the optimal temperature for S. cerevisiae culture?
30 °C
166
. 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)?
0.02 mm3
167
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?
50%
168
If you perform a 1 in 10 dilution, what’s the dilution and
dilution factor?
1/10 and 10
169
What did we do in lab 6 with RBCs?
Fractionate red blood cells and determine protein concentration
170
What are the main methods for determining protein concentration?
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
What protein standard is used to determine protein concentration?
Known concentrations of bovine serum albumin (BSA)
172
Since we can't measure BSA using spectrophotometry, what do we do?
the protein samples are mixed with a colorimetric substance --> the Bradford Reagent
173
What do we use to make s standard curve to determine protein concentration
A protein standard containing a known concentration of protein is mixed with the Bradford Reagent and the absorbance value read
174
What does the Bradford reagent contain?
Coomassie Brilliant Blue
175
What does the Bradford reagent bind to quantitively?
to arginine (basic) and aromatic amino acids such as tyrosine, phenylalanine and tryptophan
176
What wavelengths does the Bradford reagent shift from when bound
470 to 595
177
What is the max wavelength of Bradford reagent?
595 nm
178
What do we plot on the BSA standard curve?at what absorbance
absorbance at 595 nm and concentration of BSA
179
What will we use the BSA standard curve to calculate?
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
What is the BSA standard curve also known as?
Brandford assay
181
How do we isolate the plasma, lysate and membrane fraction?
differenital centrifugation
182
How do we plot the BSA standard curve?
known concentrations of bovine serum albumin to use as a protein standard.
183
What is the extracellular matrix of blood?
Plasma
184
What do RBC do in a isotonic saline?
In isotonic saline have a net zero exchange of water across the membrane.
185
What do RBC do in a hypotonic solution
have a net influx of water causing the cells to lyse
186
What is fractionation?
procedure by which cells or tissues are broken down, and their components separated using centrifugation
187
What techniques do we use for blood fractionation (2)
osmotic lysis and differential centrifugation
188
What do we separate our blood into
Plasma, lysate (RBC cytoplasm), and cell membrane
189
What is separated from the blood at low speeds?
Plasma from erythrocytes
190
What is between the plasma and RBC?
Leukocytes and platelets (Buffy coat)
191
Once erythrocytes are separated from the plasma, what do we do to them?
lysed in a hypotonic solution
192
What do we separate out after lysing the RBC using centrifugation?
Lysate and membrane
193
What causes proteins to easily degrade and what do we do to stop this
ubiquitous cellular proteases released from cellular compartments following osmotic lysis degrade proteins.
Slowed down by keeping samples cold
194
Is coomassie blue acidic or basic?
acidic
195
What do coomassie blue in the Bradford assay bind to
binds quantitatively to arginine (basic) and to aromatic amino acids such as tyrosine, phenylalanine and tryptophan residues in the proteins.
196
How long is the Bradford assay reliable for
about an hour
197
What protein standard do we use to make our standard curve in the Bradford assay?
bovine serum albumin (BSA), which is extracted from the blood of cattle
198
Why do we use BSA specifically. Could something else be used?
Used because it contains lots of arginine and aromatic amino acids
199
What is the Bradford assay?
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
What happens when the Bradford reagent (coomassie blue) binds to BSA or proteins
shifts from brown to blue
201
What did we use the Bradford assay for?
To determine the protein concentration in our membrane, lysate, and plasma samples
202
What proteins doe the plasma consist of
serum albumin, globulins, clotting and hormone-binding proteins
203
What proteins are found in the lysate?
hemoglobin
204
What proteins are found in the membrane
membrane proteins and cytoskeleton proteins
205
What do we do with the interpolated protein concentrations we measure using the equation of the line during the Bradford assay?
Multiply it by the dilution factor to get the adjusted protein concentration
206
Why do we keep centrifuging the RBC pellet wit isotonic saline?
Removes plasma proteins from the RBC
207
208
After we lyse the cells using hypotonic saline and centrifuge, what is in the supernatant and what is the pellet
lysate with hemoglobin and membrane is the pellet
209
What do we continue to wash the membrane fraction with hypotonic saline. What colour should it be when we are done?
To remove lysate proteins so they don't affect the accuracy of the protein concentration
Light pink or clear
210
When making their BSA standard curve, do we add Bradford reagent to every sample? Do we add BSA to every sample?
Yes
No, the concentrations of BSA varies
211
What absorbance do we use for the BSA standard curve?
592 nm
212
Do we use the concentration of Bradford reagent when calculating final BSA concentration? Why?
No, because each tube contains the same amount of Bradford reagent and we are interested in knowing the concentration of BSA.
213
What do we put in the BSA standard curve?
BSA protein concentration and absorbance
214
What do we have to do to our protein fraction prior to determining protein concentration? Why?
We must perform a serial dilution to the plasma and lysate because the protein concentration is too high for the standard curve.
215
What do we dilute the plasma, lysate, and membrane fraction to? What what dilution steps did we use?
Plasma 1/100
Lysate 1/1000
Membrane 1/1
1/10 with a total volume of 100 uL
216
What do we do with the membrane, plasma and lysate fractions after we dilute them? What do we use as our blank?
100 uL of hypotonic saline
Add 1000 uL of Bradford to all of our samples and let sit for 5 minutes
217
How long do we let the proteins with the Bradford reagent sit for before reading absorbance?
5 minutes
218
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)
input the absorbances into the equation of the line and solve for x. Then multiply to the dilution factor
219
Describe a suitable control to test the specificity of the Bradford assay for certain amino acids.
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
What did we do to prepare our plasma sample for SDS page? and dilute it to what?
Mix it with 2X sample buffer
1/2 dilution
221
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 ____
Into the cell
equally across the cell
out of the cell
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Protein quantification occurs when Coomassie Brilliant blue binds to any one of the known amino acids.
False
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Observe how the mixture becomes clear after the addition of the hypotonic saline. Why does the sample become more clear?
a. Cells are osmotically lysed
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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
10 uL, 90 uL, 2, 3
225
What were our protein samples mix with for SDS page?
2X sample buffer
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What does the 2X sample buffer contain?
Beta-mercaptoethanol:
Sodium dodecyl sulfate (SDS)
Bromophenol blue
Glycerol:
227
What is the function of Beta-mercaptoethanol in 2X sample buffer
denatures the protein by cleaving disulphide bonds
228
What is the function of SDS in the 2X sample buffer?
denatures and binds to proteins giving them a negative charge
229
What is the function of glycerol in 2x sample buffer?
adds density to the sample
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What is the function of Bromophenol blue in the 2x sample buffer?
tacking dye that allows us to see the sample moving through the gel
231
What is the function of the protein standard/ladder? And what does it contain? is it mixed with 2x sample buffer?
is used to estimate the approx. weights of the protein samples.
contains a mix of proteins of KNOWN molecular weight (kDa).
YEs
232
What do we use coomassie brilliant blue for after electrophoresis? and what does it bind to?
Coomassie brilliant blue dye reacts and binds to arginine and
aromatic amino acids
We stain the gel with it to reveal the proteins
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What are the two layers of an SDS-PAGE gel
Stacking and resolving gel
234
how will we the results of SDS-PAGE to determine?
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
What is electrophoresis?
a method commonly used to separate charged molecules such as proteins
236
Where will negatively charged molecules migrate during electrophoresis (no SDS)? What about positively charged?
negatively charged molecules will migrate towards the positive pole (anode) --> down
Positively charged molecules will migrate towards the negative pole (cathode)
237
Why are different bands formed in SDS-PAGE gel?
olecules that differ in charge and size will migrate to form distinct bands in a gel matrix
238
What does SDS-PAGE stand for?
sodium dodecyl sulfate polyacrylamide gel electrophoresis
239
What is SDS-PAGE used for
SDS PAGE is used to discover the molecular weights of proteins in a protein sample
240
What is the gel made out of for SDS page?
polyacrylamide gel matrix
241
What does SDS do to the proteins?
denatures and binds to proteins leading to an overall negative charge (-1 charge)
242
How much SDS binds to 1 g of protein and what do this do?
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
What is the negative charged during SDS-Page determined by?
by SDS and not by the charge of the protein
244
Why is it important that the charge-to-mass ratio is the same for SDS-PAGE
When the charge-to-mass ratio is the same, proteins will seperate based on on molecular weight.
245
What is the function of a protein ladder?
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
How do we make the standard curve of the protein standards?
We used the protein ladder and use the log of the molecular weights and the distance traveled in the gel.
247
What is the x and y value for the standard curve to determine protein molecular weights?
x= molecular weight
y = length
248
What do we do once the gel is completely stained with coomassie blue?
de-stain it using water
249
Do small or large proteins move the fastest through the gel?
Small
250
What is the function of the stacking layer?
To get all of the protein samples lined up so they can enter the resolving layer at exactly the same time
251
What is the function of the running buffer in the tetra cell?
allows for conductivity
252
What protein samples did we test in SDS page?
membrane,
Plasma
Protein A or B
253
Does the resolving layer contain more acrylamide?
yes
254
What does the TMED and APS do when you add it to the gel layers
polymerizes them
255
What way does the small plate face when making a SDS gel
you
256
What is our standard curve of protein weights based on?
tandard curve of the log10 of molecular weights of the protein standard against the distance traveled from the top of the resolving layer.
257
What do we do with our protein standard curve for molecular weight?
Input the length of one of our sample for y and find X
258
Once you find the value X, the molecular weight, what do you have to do
10^x
259
What do do if spills are noxious or irrittating?
evacuate
260
What to do with a spill of microorganisms
dilute the spill with a 10% solution of bleach
261
If there is a minor fire what do you do
call for help, attempt to control it, then if it isn't controlled in 1 minute leave
262
What are type A extinguishers used for
ONLY for trash, wood or paper combustibles.
263
What are type B extinguishers used for?
ONLY grease or liquids
264
What are type C fire extinguishers used for
ONLY electrical fires
265
What are type ABC extinguishers used for
all types
266
What are type BC fire extinguishers used for?
electrical, oil, grease, liquid
267
What is WHMIS?
Workplace Hazardous Materials Information System
268
What are the four components of WHMIS
Product Labeling
Labware labeling
Material Safety Data Sheets (MSDSs)
WHMIS Education and Training Programs
269
WHAT IS THIS?
Compressed gas
270
What is this
flammable or combustible materials?
271
What is this? and what does it do
oxidizing materials
Increases risk of fire if they come in contact with flammable or combustion able materials
272
What is this?
materials causing serious toxic effects. may cause death in small amounts
273
What is this? What does it mean
Materials causing other toxic effects?
can cause long term health effects
asbestos
274
What is this
Biohazardous infectious materials
275
What is this
Corrosive materials. Products which may self-react upon standing.
276
Where will a positively charged protein migrate in SDS page
A positively charged protein in SDS page will become negative due to the SDS and will migrate to the anode.
