Exam II Flashcards
1
Q
What are the steps of gene cloning?
A
- Isolation of plasmid DNA and DNA containing gene of interest
- Gene inserted into plasmid
- Plasmid put into bacterial cell
- Cells cloned with gene of interest
- Identification of desired clone
2
Q
What was our gene of interest that we cloned using TOPO?
A
adhP
3
Q
What does the gene adhP encode for?
A
Alcohol dehydrogenase
4
Q
What method is used to get the plasmid back into a host cell?
A
Transformation
5
Q
What is the plasmid we used in lab called?
A
pET-101D/TOPO
6
Q
How will we purifiy our cloned proteins in lab?
A
Affinity Chromatography
7
Q
What does PCR stand for?
A
polymerase chain reaction
8
Q
What are two ways cloning can be achieved?
A
- take a small portion of organisms and allowing it to grow into a full organism (carrot)
- Embryo Splitting
9
Q
What is a fundamental process of copying the DNA which occurs in all living organisms and is the basis for biological inheritance?
A
DNA replication
10
Q
What is the difference between DNA and RNA?
A
- DNA is double stranded, with two anti-parallel strands
2. RNA is a single strand and can form secondary and tertiary structures (tRNA)
11
Q
What are the components of a nucleotide?
A
Base
Sugar
Phosphate
12
Q
The base can either be a purine or a what?
A
pyrimidine
13
Q
Adenine and guanine are what type of base?
A
purine
14
Q
Thymine/Uracil and cytosine are what type of base?
A
pyrimidine
15
Q
Guanine and cytosine form how many hydrogen bonds?
A
3
16
Q
Thymine/Uracil and adenine form how many hydrogen bonds?
A
2
17
Q
The side chain group on the 2’ Carbon on DNA is what?
A
H
18
Q
The side chain group on the 2’ Carbon on RNA is what?
A
OH
19
Q
What group is on the 3’ Carbon for both RNA and DNA?
A
OH
20
Q
The phosphate backbone attaches to which carbons of both DNA and RNA?
A
5’
3’
21
Q
What is ligation?
A
When a phosphate group attaches to the 3’ or 5’ carbon of a sugar
22
Q
What serves as the template for the reproduction of the complementary strand?
A
Each strand of the original double stranded DNA molecule
23
Q
What are produced from a single double stranded DNA molecule?
A
Two identical DNA molecules
24
Q
Which strand is read 3’ to 5’ by the DNA polymerase and produces a continuously growing strand?
A
Leading strand
25
Which strand is read from 3' to 5' but produces short strands that must be linked together?
Lagging Strand
26
What is located at the replication fork and separates the lagging and leading strands?
Helicase
27
Who discovered DNA polymerase in 1956?
Arthur Kornberg (Nobel Prize in 1959)
28
What can only add nucleotides to the 3' end of the primer- extending the chain in the 5' to 3' direction?
DNA polymerase
29
What is the error rate of DNA polymerase?
1 in every billion base pair added
30
What are they short strands that are formed from the lagging strand called?
Okazaki fragments
31
PCR is a process that imitates what?
the natural DNA replication
32
The membranes of bacterial cells are made up of what?
lipids
33
The cell walls of bacterial cells are made up of what?
sugars
34
What kind of bacteria has both an inner and outer cell wall?
Gram (-) Bacteria
35
Gram positive bacteria have a thick layer of what?
peptidoglycan
36
Gram negative bacteria have what kind of peptidoglycan layer?
thin
37
What are plasmids?
Non-chromosomal DNA
38
Does the replication of plasmids depend on the replication of the chromosome?
No
39
Are there many copies of plasmids within a cell?
Yes
40
What are the steps of genomic DNA purification?
1. Lyse all cells
2. Denature proteins
3. Inactivate endogenous nucleases
4. Isolate/ Purify genomic DNA
41
What does Tide Free 2X laundry detergent contain?
proteases
lipases
glycolytic enzymes
42
What is the function of the proteases in Tide?
degrades proteins
43
What is the function of the lipases in Tide?
cleaves fat/lipids
44
What is the function of the glycolytic enzymes in Tide?
degrade sugar
45
What has limited solubility in water; thus forms a separate organic phase from the aqueous phase?
n-butonal
46
What do you do to separate the cell debris, organic phase, and inorganic phase?
Spin the sample
47
Which phase is removed and transferred to a new tube after the n-butanol is added and the tube is spun?
The aqueous phase (inorganic)
48
Which phase will the DNA and RNA go to and why?
aqueous (inorganic). They are starches
49
Why do you add 2-propanol to the aqueous layer?
To precipitate the genomic DNA and RNA
50
Why do you wash with 70% ethanol?
To remove salt contamination
51
why is it crucial that you dry out the ethanol before re-dissolving the DNA pellet back in water?
maintains the activity of enzymes used further down in the protocol.
52
What is added to remove RNA contamination?
RNase A (incubate at 60 C)
53
What are the components of PCR?
```
two primer strands (forward and reverse)
dNTPs
template DNA
appropriate buffer
DNA polymerase
```
54
Who invented PCR?
Kary Mullis (nobel Prize 1993)
55
What are other names for the coding strand?
sense strand
| positive strand
56
What are other names for the complementary strand?
anti-sense strand
| negative strand
57
What should be the length of the primers?
20 to 30 nucleotides long
58
What is the denaturation step of PCR?
DNA is heated to separate strands 95 C
59
What is the annealing step of PCR?
DNA strands are cooled to allow them to anneal with the primers (52-65 C)
60
What is the extension step of PCR?
Temperature is increased to ~72 C and DNA Polymerase completes the DNA strand
61
What direction does synthesis/replication occur?
5' to 3'
62
How many cycles can a PCR reaction run for?
25-35 cycles
63
A PCR reaction with N cycles will give how many DNA molecules?
2^N
64
What has the same sequence as the "coding" strand and anneals with the complement strand?
FWD primer
65
What has the same sequence as the complement strand and anneals with the coding strand?
REV primer
66
What determines the specificity and significantly affects the ability for the primer to anneal to the template?
primer length
67
If the primer is too short what occurs?
low specificity, resulting in non-specific amplification
68
If the primer is too long what occurs?
decreases the template-binding efficiency at normal annealing temperatures due to the higher probability of forming secondary structures such as hairpins
69
What are some examples of secondary structures?
hairpins
self-Dimer
Dimer
70
How do you determine the annealing temperature?
Should be a few degrees lower than the melting temperatures of the primers which is based on the number of GC and AT pairs
71
What is the formula to estimate the Tm?
Tm = 4 (G+C) + 2 (A+T) units are in degrees Celsius
72
What is a device that controls incubation temperatures and times?
Thermal Cycler
73
For how long is the denaturing step?
15 to 30 seconds
74
How long is the elongation step?
1 to 2 min for every 1000 base pairs
| 1 min/kb at 68 to 74 C is optimal
75
How long is the elongation step?
5 to 10 min at 68 to 72 C
76
At what temperature should you store the genes?
4 C
77
When does the product that consists only of the desired regions of DNA appear?
3rd cycle
78
Each step is what, since two strands coming from the newly synthesized DNA can act as a template for the next reaction?
exponential
79
Does each strand need one primer, with opposite polarity?
Yes
80
What should be the ratio of absorption at 260 and absorption at 280 for DNA and proteins?
DNA 2
| protein 0.57
81
What is the extension coefficient for ds DNA at 260nm?
50 micrograms/mL
82
What is the extension coefficient for ssDNA at 260 nm?
33 micrograms/mL
83
How long is the annealing step?
15 to 30 seconds
84
How do you calculate the number of DNA copies that span the size between the primers after n cycles of PCR?
2^n - (n*2)
85
What matrix did we use in the gel electrophoresis?
0.8% agarose
86
Agarose gel electrophoresis separates molecules based on what?
size, shape, and charge
87
What charge does DNA have?
negative
88
Do smaller or larger molecules move faster in gel electrophoresis?
smaller
89
negatively charge DNA moves toward what?
the anode
90
molecular size markers contain what?
different DNA fragments of known size
91
What buffer is usually used in electrophoresis?
Tris-acetate-EDTA (TAE)
92
The loading buffer contains what which allows the sample to fall into the sample wells?
something dense like glycerol
93
The loading buffer also contains this allowing for the visual monitoring or how far the electrophoresis has proceeded?
one or two tracking dyes
94
How do you pour a gel?
```
weigh 0.8% agarose
add electrophresis buffer
microwave until completely melted
cool to 60 C
add fluorescent stain
pour into casting tray containing comb
allow to solidify
```
95
DNA will migrate toward the positive what?
electrode (anode)
96
What is intercalating mean?
inserts between the bases of DNA
97
what kind of dye is used?
fluorescent intercalating
98
What are some examples of the dye used?
SYBR safe or Ethidium Bromide
99
SYBR safe is how many times less toxic than Ethidium Bromide?
4 - 5 times
100
Do supercoiled (uncut), relaxed circular form, or linearized forms of plasmids travel the fastest?
supercoiled
101
Which form of plasmid travels the slowest?
relaxed circular form
102
What migrate through agarose gels with mobility that is inversely proportional to the log base 10 of their molecular weight?
linear DNA
relative mobility = 1/(log MW)
103
Typically the size of DNA bands are reported as the number of what?
base pairs or kilo base pairs
104
Which resolves better at low % gel, larger or smaller DNA?
larger 0.7%
105
Which resolves better at in 1.5% agarose, larger or smaller DNA?
smaller
106
What migrate proportionally faster as the voltage applied to a gel is increased?
larger DNA fragments
107
What is the best resolution of fragments that are greater than 2 kb?
5 V/cm
108
What functions do the electrophoresis buffer have?
establish pH
provide ions to support conductivity
maintain DNA
109
What are the potential reasons that a PCR does not work?
1 old/dead polymerase
2 wrong/degraded primers
3 too high concentration of primers or templates
4 wrong annealing temperature
5 ethanol (kills DNA polymerase) contamination of template DNA
110
annealing temperatures that are too low can result in what?
non specific priming which can lead to smears in electrophoresis
111
What is the start codon for E.coli?
ATG
112
What are the stop codons?
TAA, TAG, and TGA
113
What two amino acids have only one codon?
Met (ATG) and Trp (TGG)
114
Where can you retrieve the gene sequence?
NCBI (National Center for Biotechnology Information)
115
What is an accession number?
A unique identification number of a sequence. It is a string of letters and/or numbers that corresponds to a molecular sequence (protein or gen sequence)
116
What button to you select to retrieve the gene sequence?
FASTA
117
What is a short synthetic oligonucleotide designed to have a sequence which is the reverse complement of a region of template or target DNA?
a primer
118
The primer must have what to prevent fraying
A GC clamp
119
What is the temperature at which one half of the DNA duplex is a stable double helix, while the other half has been separated to single strand molecules?
Melting temperature
120
What is the optimum difference in the melting temperature of primers?
< 5 C (actually 3-5)
121
IDT has what tool to analyze primer?
Oligo analyzer
122
In addition to the melting temperature what else should be analzyed?
whether secondary structures are made.
123
What are modified plasmids used in recombinant DNA for cloning?
Vectors
124
What must vectors have?
ORI (origin of replication)
a drug resistance gene (reporter gene)
a Multiple Cloning Site (MCS)
125
What is a MCS?
a region in which exogenous DNA fragments can be inserted
126
TOPO cloning uses DNA topoisomerase for what?
directed cloning
127
TA cloning uses Taq polymerase that does what?
Adds "A" at the 3' end of the PCR product and is non-directional
128
Restriction Enzyme mediated cloning produces what two ends?
blunt (non-directional)
| sticky (directional)
129
What is an advanced method where there is no need to use restriction enzymes to produce directional clong?
Ligation Independent Cloning
130
What antibiotic resistance gene does pET101D have?
ampicillian
131
What two sequences does pET101D have that are essential for T7 RNA polymerase to transcribe the gene?
T7 promoter and T7 terminator
132
What is LacO?
an operator sequence (regulatory) that controls the protein expression
133
What is LacI
binds to LacO and prevent activity of T7 RNA polymerase (suppressing) expression of recombinant protein.
134
What is IPTG?
a lactose analog that binds to LacI allowing LacI to leave the operator
135
What leads to a better yield and solubility of the recombinant protein?
controlled expression
136
What is the function of V5 eptitope?
codes for an I4 aa peptide
137
What is used for convenient detection by recognizing the I4 aa peptide?
Western blot
138
pET101D has a 6xHis tag at the what of the protein of interest?
C-terminus
139
When using his tags, the primers should not contain what?
stop codons
140
What kind of columns can bind to the his tag and purify the protein of interest?
Ni-NTA columns
141
What is the RBS?
ribosome binding site
142
What is the TOPO site?
where DNA topisomerase is covalently attached
143
Where is the STOP codon on the pET101D vector?
after the 6xHis and V5 eptitope
144
DNA topoisomerase I recognizes what, makes a cut, and then remains attached to the phosphate of the nucleotide?
CCTT sequence
145
The 5' end of the PCR product will have a region that matches the overhang with what sequence?
GTGG
146
The topoisomerase promotes what?
the ligation of the vector and the PCR product
147
Review slide 24 Lecture 13
Review it!
148
What are some advantages of TOPO cloning?
1. does not require restriction enzymes
2. directional insertion
3. doesnt require ligation
4. less time required for procedure
149
What are some disadvantages of TOPO cloning?
1. The overhangs in the vector are sensitive to degradation
| 2. have to purchase vector every time from the vendor (can be costly)
150
What is essential for T7 RNA polymerase to transcribe the gene?
T7 promoter and T7 terminator sequences
151
What are endonucleases that cleave DNA strands?
Restriction enzymes
152
What provides a defense mechanism against invading viruses?
Restriction enzymes
153
Over 3000 what have been studied?
Restriction Enzymes
154
In many cases the restriction site is what?
self complementary (palindromic)
155
What happens when pET101D + adhP is cut with SacI
a single strand is formed
156
What happens when pET101D + adhP is cut with PstI?
two strands are formed
157
What kind of ends does SmaI create?
blunt ends
158
What kind of ends do both EcoRI and BamHI form?
staggered or sticky end cuts
159
What covalently attaches the vector and the insert via a phosphodiester bond between 5' phosphate and 3' hydroxyl?
ligation
160
After deciding which vector you will be using, you choose two different RE sites that are not what?
present within your gene of interest
161
Sequences must be in what?
frame
162
When you have an N-terminal His tag where do you insert the gene?
after the His-tag
163
Inserted junk is usually what size?
5 to 6 nucleotides
164
What is order of information added to fwd primer in enzyme restriction?
5' Junk ,Restriction Enzyme, gene we want minus the beginning methionine 3'
165
What is the order of information added to make the reverse primer using enzyme restriction?
5' Junk, restriction enzyme, end of gene with stop codon (will be in reverse) 3'
166
Which method of restriction enzyme cloning is not efficient, non-directional, has high rates of self-ligation and is time consuming?
Blunt ended cloning
167
Which method of restriction enzyme cloning requires restriction digestion of insert and vector (RE should be absent internally in the insert), has two different sticky ends, directional insertion, high ligation efficiency, but is time consuming?
sticky ended cloning
168
in oligo design the tail should have what beyond the restriction site for the enzyme to grab onto?
5 to 8 bp
169
The reverse primer may need to encode what?
a stop codon
170
NdeI and Ncol encode what in their restriction sites?
ATG
171
What are the mechanisms of DNA transfer?
Conjugation
Transduction
Transformation
172
What is conjugation?
The physical interaction between cells with a direct transfer of DNA from one bacterium to another
173
What is transduction?
Virus mediated transfer of DNA between bacteria
174
What is transformation?
When DNA is taken up from the environment by bacteria
175
What are bacterial cells that are able to take up DNA from the environment called?
Competent cells
176
Why do competent cells carry?
Genes that encode proteins called competence factors
177
The process of making a cell competent usually involves what?
Ca++ that shields the negative charge of DNA
178
Top10 competent E.coli cells do not contain what?
T7 RNA polymerase
179
When you are ready to perform an expression experiment you should transform your construct into what?
BL21 Star (DE3) E. Coli
180
Why do we use a restriction site in the insert during Top10 cloning?
To determine if the gene of interest is present. An additional strand will be present because of an additional cut.
181
During plasmid purification, what happens to the genomic DNA and RNA?
DNA is excluded because it is larger and more supercooled, RNA is degraded
182
What are the basic steps of Plasmid DNA purification?
1. Pellet the cells
2. Resuspend in a neutral buffer containing RNAse
3. Lyse the cells
4. Denature proteins and genomic DNA
5. Capture plasmid DNA
6. Wash t remove excess salt
7. Elute plasmid DNA
183
An alkaline solution is used to do what to cells?
Lyse
184
What components are included in the lyse solution?
```
SDS detergent (a detergent)
Sodium Hydroxide (a base)
```
185
Why is a basic pH used for the lyse step?
It denatures the dsDNA into single strands
186
What does SDS do?
Denature proteins
187
How is the solution neutralized during plasmid purification?
Adding sodium acetate pH 5.5 that includes chaotropes which the proteins. As the pH drops plasmid DNA will renature due to its small and circular nature.
188
Why do we not use vortexing?
To prevent the shearing of genomic DNA
189
What happens to the DNA and proteins during the neutralizing step of plasmid purification?
They clump together and fall out of solution
190
How do we capture the plasmid DNA in plasmid purification?
Option 1 ethanol precipitation
Option 2 bind the DNA to a Silva membrane - binds DNA at optimal pH and salt concentration - this can be washed with ethanol
191
Why do we wash the silica membrane with ethanol?
The high salt is removed by the 70% ethanol wash, while DNA is bound to the membrane
192
How is the plasmid eluded off the silica membrane
Using water or Tris buffer with no salt by applying it directly to the membrane followed by a short incubation spin
193
How can the purity of the DNA be confirmed?
Ratio of 260/280 absorption’s
194
What are the steps for agarose gel electrophoresis?
1. Load sample
2. Application of electrical field (DC)
3. DNA separation
4. UV transillumination and documentation
195
Minipreps produce what kind of DNA?
Supercoiled DNA with trace amounts of nicked DNA
196
What does BL21 Star (DE3) have that promotes expression?
Lambda DE3 lysogen which carries the gene for T7 RNA polymerase under the control of the lacUV5 promoter
197
What is required to induce the expression of the T7 RNA polymerase?
IPTG
198
BL21 Star (DE3) carries a mutated Rne gene which does what?
Encodes a truncated RNAse E enzyme that lacks the ability to degrade mRNA resulting in increased mRNA stability
199
Translation will only start at what codon?
AUG/ATG
200
What program do we use to find the pI or translate the gene?
Expasy
201
BL21 Star does not contain the Ion protease and has a mutation in the outer membrane protease, Omp T. Why?
Reduces degradation of heterologous proteins expressed.
202
Neutralizing the pH contains sodium acetate and guanidium chloride that do what?
Denatures proteins
203
What are two types of sources for proteins?
Natural
- organism
- tissue
- growth stage
Recombinant
- gene sequence
- design primers, PCR
- clone (vector choice)
- host cell
- express
- purify
204
What are the molecular characteristics of your protein?
```
Charge
Hydrophobicity
Solubility
Stability
Size
Ligand
Structural Integrity
```
205
What two types of expression are there?
Intracellular and extracellular
206
Is our protein soluble in cytoplasm?
Yes
207
What are ways to lyse cells without damaging the protein?
```
Mechanical disruption - blender
French Press
Sonic action
Freeze Thaw Cycles
Enzymatic
```
208
How does lysozyme work?
Cleaves the peptidoglycan bonds and degrades the cell wall
209
What are detergents?
Compounds with hydrophobic and hydrophilic ends, they will integrate into membranes and dis-integrate the membrane. Useful for gram-negative bacteria since they have an outer membrane over the thin layer of cell wall
210
Why do we add PMSF?
Phenylmethylsulfonylfluoride inhibits protease
211
What temperatures do you want to work at?
Lower 4 C
212
Can you protect your protein from degradation by using special expression cells?
Yes
213
What do you centrifuge for protein purification at?
30,000 G for 30 minutes to an hour
214
The concentration of any salt necessary to cause precipitation of a particular protein is related to what?
The number and distribution of charges and of hydrophobic residues exposed.
215
As salt concentration increases, protein solubility does what?
Decrease
216
Middle stages of purification take advantage of what?
Properties of the protein
217
What is based on the surface hydrophobic amino acids’ components of proteins?
Hydrophobic Interaction Chromatography
218
Ion-Exchange chromatography takes advantage of what?
The pI of your protein
219
Affinity chromatography takes advantage of what?
Resins contains ligand or substrate covalently attached and is used with protein affinity tag (His-tag)
220
Where do you get the gene sequence from?
NCBI
221
How do you translate it to protein (the gene)?
Expasy
222
To find the pI of your protein what do you add to your gene?
V5 and his-take
223
What methods are used to capture proteins that have an affinity tag?
Affinity chromatography
224
What methods are used to capture proteins without an affinity tag?
Ion exchange and HIC (hydrophobic interaction chromatography)
225
What is an intermediate step in protein purification with an affinity tag?
Ion exchange chromatography
226
What is an intermediate step for protein purification without an affinity tag?
HIC or IEX
227
What method is used to polish the protein whether it has an affinity tag or not?
Gel Filtration
228
After reaching what optical density, the protein was expressed by the addition of 1 mM IPTG
OD600 of ~0.6
229
What is IPTG?
A lactose analog
230
Histidine tag coordinates with metals like what?
Ni and Cu
231
What is used to elute a protein from an affinity column?
Imidazole
232
What else can be used to elute a protein from an affinity column?
Engineered protease site
233
What are other names of affinity chromatography involving metals?
Metal chelation chromatography or immobilized metal affinity chromatography (IMAC)
234
In IMAC what are the resins made with?
Nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA)
235
What is the pKa of his?
~6.5
236
What is the role of the low concentration of imidazole in the lysis and wash buffers?
To elute out proteins with a few histidine
237
What are some polishing methods?
IEX, GF, ultra-filtration/centrifugal concentrators
238
In dialysis smaller molecules can move in and out until equilibrium is reached but it is important that what is selected?
The right size of membrane
239
What labs were performed for AdhP expression and purification?
Cloned into pET101D vector with 6xhis tag
Transformed into IshotToP10 plasmid prep
Plasmid was analyzed by RE digestion
Transformed into BL21DE3
Expressed by addition of IPTG
Harvested Cells lyse and purified via Ni-NTA affinity chromatography
240
What is the pH of the running buffer?
8.3
241
What is the percentage and pH of the stacking gel?
4% pH 6.8
242
What is the percentage and pH of the resolving gel?
7 to 15% pH 8.8
243
SDS Page Theory involves what?
The movement of charge particles under the influence of an electric field
244
Negatively charge molecules move toward what?
The positive anode
245
Positively charged molecules move toward what?
The negatively charged cathode
246
What is the reaction for anode (oxidation)?
H20 -> 2 H + 1/2 O2 + 2 e
247
What is the reaction for cathode (reduction)?
2e + 2H2O -> 2 OH + H2
248
How many more bubbles arise from the cathode?
Twice as many
249
The pH does what in the cathode chamber?
Increases
250
The pH does what in the anode chamber
Decreases
251
Acrylamide is a dangerous what?
Neurotoxin
252
What is TEMED used for?
Helps cleave persulfate
253
The free radical (sulfate radical) does what?
Forms crosslinkages between N-methylenebisacrylamide (monomer)
254
Acrylamide solutions come in what ratios?
19:1, 29:1, 37.5:1
Ex for every 19 acrylamide there is 1 bisacrylamide
255
SDS coats to a density of what?
1.4g SDS per g polypeptide
256
What is SDS used for?
Denatures protein and gives it a negative charge (protein is effected by differences in size only)
257
Reducing agents like dithiothreitol (DTT) do what?
Break covalent disulfide bonds (cystines) and converts into monomers
258
what happens in the stacking gel?
Proteins are concentrated as a thin band at the interface
259
What happens in the resolving gel?
Proteins get separated based on size
260
What should the properties of the loading dye be?
Viscous
Color to track run
SDS and reducing agent
PH of 6.8
261
Who developed the SDS-Page?
Ulrich Laemmli
262
SDS-Page is called discontinuous electrophoresis because of what?
The pH differences from 8.3 to 6.8 to 8.8
263
What charge does the protein have in the running buffer?
Negative
264
What charge do the proteins have in the stacking gel?
Neutral
265
What charge do the proteins have in the resolving gel?
Negative
266
The charge distribution is based off what amino acid?
Glycine
267
The fast moving Cl- ions and mostly neutral glycine molecules create what?
A zone of low conductance or high resistance
268
At constant current V=IR, the high resistance leads to what?
A localized increase in the electric field
269
For SDS mobility is directly proportional to what?
The charge of the molecule and is inversely proportional to the frictional coefficient
270
When denatured by heat and coated with SDS, what becomes nearly the same for all polypeptides?
Shape and charge
271
Coomassie Brillant blue R 250 (different from dye used in Bradford assay G-250) has what charge so that it will bind to the protein?
Positive
272
What is the equation for relative mobility?
Rm = distance migrated by protein/distance migrated by the dye front
273
How can you determine the MW of an unknown sample
Plotting log of known molecular weights with relative mobility
274
One unit of enzyme is defined how?
The amount of enzyme required to generate 1 micromole of product in one minute at a given pH and temperature (Standard 25 C or 37 C)
275
Activity coeffiecient for NAD+ at 260 is what?
18,000
276
Activity coefficient for NADH at 260 is what?
15,000
277
Activity coefficient for NADH at 339?
6,220
278
Activity coeffiecient for NAD+ at 339 N.M.?
0
279
What does AdhP do?
Catalyzes the oxidation of alcohol to aldehyde in a zinc dependent manner in the presence of cofactors NAD+ and NAD+ get reduced to NADH (absorbs at 340)
280
Activity is express how?
As micromoles of product formed per min
281
How do you determine total activity?
Units*ml
282
How do determine specific activity?
Divide the activity (units) by protein (mg)
283
How to you determine the fold of purification?
Divide the specific activity by the specific activity of the Lysate
284
How do you determine % yield?
(Total activity/total activity of lysate)* 100
285
In IEF (isoelectric focusing) Gel the proteins stop moving when what?
The net charge of the protein is zero.
286
Isoelectric focusing separates proteins based on what?
Their pI
287
What is 2D electrophoresis?
Separates first by pI and second based on size
288
What technique is used to detect the presence of a specific protein in a mixture to confirm the identity of a particular protein?
Western Blot
289
What does the Western blot take advantage of?
The highly specific interaction of antigen to an antibody
290
What is an antigen?
A molecule that can induce an immune response
291
What is an antibody?
A Protein produced by specialized white blood cells to recognize and inactivate an antigen
292
What is an epitope?
A portion of an antigen that is recognized by the antibody
293
What are steps of Western Blot?
Separate on SDS page
Transferred to a positively charged membrane
Bound by antibodies
Detected by reaction - the interaction between the antigen and antibody is detected by a colorimetric or fluorescent reaction
294
Western Blot overview steps are what?
```
SDS-Page
Transfer
Block
Rinse
Antibody
Rinse
Color Development
```
295
What is the difference between nitrocellulose membranes and polyvinylidene difluoride membranes?
Nitrocellulose are excellent protein binding and retention capabilities but are brittle and not used when proteins need to be stored
(PVDF) have superior mechanical strength making it suitable for stripping/reproving and for further characterization techniques such as sequencing and proteolytic. Take care when staining
296
What is the fragment antigen binding?
A region on an antibody that binds to antigens
297
The fragment constant (Fc) composition will be what for all antibodies produced in an organism?
Identical
298
What is the difference between monoclonal and polyclonal antibodies?
Monoclonal
Specificity for a single epitope
Less sensitive since only a single antibody molecule binds to each target
Polyclonal
Varying specificities to multiple epitopes
More sensitive
299
There are how many antibodies involved in Western blotting?
Two , primary, secondary - enzyme conjugated
300
In our experiments the primary antibody is directly conjugated to what?
The HRP enzyme
301
What is a mouse monoclonal IgG2a antibody that is itself conjugated to an enzyme?
Anti-V5-HRP the enzyme is horse radish peroxidase (HRP)
302
A chromogenic substrate is added that HRP will catalyze to produce what?
Color
303
HRP is a heme containing oxidoreductases enzyme that catalyzes the oxidation of 4-Chloro-I-naphthol (4CN) in the presence of what?
Hydrogen peroxide
304
what is the product formed by HRP?
An insoluble purple quinone
305
When you want to clone a gene into a TA vector, what DNA polymerase should you use in the PCR and why?
Taq Polymerase it adds a A at the end of the 3’ end of the PCR product so that it can be inserted directionally into a TA vector.
306
When you want to clone a gene into a TOPO vector, what DNA polymerase should you use in the PCR and why?
DNA topoisomerase because it will insert the PCR product in the correct direction
307
What is the T7 promoter and T7 terminator sequence
T7 promoter, lacO, Xba 1, RBS, Topoisomerase polymerase sites, Sac I, BstB I, V5 epitope, 6xHis, Stop,T7 terminal
