Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

Chem 474 Biochemistry l > Chapter 4 Protein Methods > Flashcards

Chapter 4 Protein Methods Flashcards

1
Q

What should protein purification yield? How does it do this?

A

Sample yields one type of molecule through using an assay
- assay: tests for unique identifying property of protein

protein should go thru series of separations based on physical properties (size and charge) to purify
- the more specific the assay, the more effective the purification

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

How do we analyze a purification scheme?

A
  • must know the amount of total protein in mixture
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Specific activity

A

ratio of enzyme activity to amount of protein in mixture

total activity / total protein

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Purification level

A

Measure of the increase in purity
- specific activity / initial specific activity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Yield

A

Measure of activity retained after each purification step
- activity in initial extract = 100%
- total activity / total activity of initial

measured in %

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is the overall goal of protein purification?

A

Maximizing the specific activity

pure enzyme = constant specific activity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What must occur for a protein to be purified?

A

The protein must be released from the cell through a series of steps

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Steps of protein purification

A
  1. disrupt cell membranes of intact cells to form a homogenate
  2. centrifuge homogenate at a low speed —> pellet forms consisting of heavy material and lighter supernatant
  3. centrifuge supernatant at higher centrifuge force —> another pellet and supernatant formed (process = differential centrifugation)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the purpose of differential centrifugation?

A

To separate the contents of a cell based on density

diff centrifugation = centrifuging a supernatant (formed from previous centrifugation) at greater forces

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Will the denser material of a cell form at a lower or higher centrifugation force?

A

A lower force
- nucleus is large + dense and will form at a lower centriguation force
- nucleus –> mitochondria –> microsomal

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What does dialysis do?

A

Separates protein from small molecules

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How does dialysis work?

A

A semipermeable membrane is used –> molecules larger than pore diameter stays in bag whereas smaller molecules diffuse down concentration gradient (into solution out of bag)

ex. semipermeable membrane = cellulose membrane with pores

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is dialysis useful for and not useful for?

A

Useful for removing a salt or small molecule from a protein, but NOT good at distinguishing between proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What causes the chromatographic process to occur?

A

Differences in the distribution constant of the individual samples

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is gel-filtration chromatography?

A

Separates proteins based on size

aka molecular exclusion chromatography

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How does gel-filtration chromatography work?

A

A column is filled with porous beads
- smaller beads enter the bead and exit last
- larger beads cannot enter the bead and flow thru –> exit first

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What is ion-exchange chromatography?

A

Separates proteins on the basis of charge

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

How does ion-exchange chromatography work?

A

A column is filled w/ charged beads
- proteins w/ same charge of bead pass thru and exit quickly
- proteins w/ opposite charge of beads will bind to the beads

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Cation exchange chromatography

A

Uses negatively-charged (anionic) beads to bind/separate positively-charged (cationic) protein

column = (-) charged carboxymethyl (CM) group

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Anion exchange chromatography

A

Uses positively-charged (cationic) beads to separate out negatively-charged (anionic) proteins

column = (+) charged diethylaminoethyl (DEAE) group

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

How can a bound protein be released from beads in ion-exchange chromatography?

A

In cation exchange, increasing the salt concentration will cause sodium ions to compete with the (+) cationic proteins and bind to the (-) anionic beads instead.

Will release (+) charged bound proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What is affinity chromatography? How does it work?

A

A column is filled with beads which are attached to a specific ligand that a protein has a high affinity for. Thus, it retains that protein.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

How is a bound protein released in affinity chromatography?

A

By passing a solution that is enriched in the ligand –> bound protein binds to solution and washes out the column

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Example of affinity chromatography

A

Concanavalin A has a high affinity for glucose. Glucose residues are placed on beads and concanavalin A will bind to the beads.
- to remove concanavalin A, high concentration of glucose is added into the column and binds to concanvalin A = releases it

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
How can affinity chromatography be utilized for proteins that do not have an identified ligand?
Through recombinant DNA technology - the desired protein can be attached to a polypeptide that binds to a highly specific, known ligand --> desired protein captured when passed through bead column
26
Example of recombinant DNA technology usage in affinity chromatography
The desired protein (unknown ligand) can be bound to a His tag, which binds tightly to nickel - run through column --> protein attached to His tag will bind to the nickel on the beads | His tag = repeats of histidine
27
Advantages in using recombinant DNA technology for protein purification
- proteins can be expressed in large quanitities - proteins can be modified with affinity tags for purification or visualization - proteins with modified primary structure can be generated
28
Histidine6
His-tag protein that was engineered by molecular cloning
29
How can GFP be purified? | green fluorenscent protein
Through hydrophobic interaction chromatography (HIC) -
30
What is electrophoresis?
The phenomenon of how molecules with a net charge will move in an electric field - used to separate mixtures of molecules with a net charge by applying an electric field
31
Direction of electrophoresis
Proteins move from negative to positive electrodes (top to bottom)
32
What is gel electrophoresis performed in?
Polyacrylamide gel
33
SDS-PAGE
sodium dodecyl sulfate- polyacrylamide gel electrophoresis - allows accurate determination of mass
34
What is SDS?
sodium dodecyl sulfate - anionic detergent that denatures proteins
35
How are proteins separated on the basis of mass?
Through SDS-PAGE
36
What is the charge on the SDS protein complex?
Large net negative charge that is proportional to the mass of protein - SDS binding is negative + greater than charge on protein
37
How many amino acids can 1 anion molecule of SDS bind to?
2 amino acids
38
How are proteins visualized after separation by SDS-PAGE?
By staining the gel with dyes - ex. Coomassie blue
39
How can electrophoresis determine protein mass?
electrophoretic mobility of proteins in SDS-PAGE is linearly proportional to the logarithm of their mass - larger mass = less mobility | figure 4.10 ## Footnote One lane is used as a marker with known molecular masses --> used to estimate the sizes of other bands
40
How can the process of protein purification be monitored?
By analyzing each fraction (of chromatography tests) with SDS-PAGE - number of bands should decrease as purification progresses until one is most prominent = protein of interest
41
Isoelectric point (pI)
the pH at which a protein has no net charge
42
Isoelectric focusing
Separates proteins in a gel on the basis of their pI - gel has pH gradient --> each protein will migrate to its pI when voltage is applied | electrophoretic mobility of a protein at it's pI = 0 bc it stops moving
43
How do you calculate the pI of a molecule/protein?
By calculating the average of it's pKas | ex. alanine has pka of 9.9 and 2.3 --> find average for pI
44
2 dimensional electrophoresis
Separates proteins in 2 directions - protein is first subjected to isoelectric focusing in horizontal direction - then SDS-PAGE in vertical direction
45
What is the result of two dimensional electrophoresis?
A gel with proteins that have been separated horizontally based on pI and vertically based on mass
46
What can two-dimensional electrophoresis detect? | what is the goal?
It can detect differences in protein expreession under diff. physiological circumstances ## Footnote - spots can be examined and differences indicate that the protein concentration changed in response to the physiological state
47
Epitope
a specific group or clusters of aa on the target molecule that an antibody recognizes | aka antigenic determinant
48
Indirect ELISA | Enzyme-linked immunosorbent assay
detects presence of antibodies - viral core proteins at bottom of well --> sample of antibodies from person added to well --> enzyme-linked antibodies added --> unbound antibodies washed off --> substrate added --> color appears if antibody is present for the antigen
49
Sandwich ELISA
Detects presence of antigen - antibody added to well --> antigen solution added and binds to antibody --> enzyme-linked antibody added and binds to immobilized antigen --> substrate added and color appears | no color if antigen isnt present bc enzyme-linked ab wont bind
50
Is the rate of color formation proportional or unproportional to the amount of antigen in sandwich ELISA?
Proportional | unproportional in indirect ELISA
51
Western blotting
separates proteins in SDS-PAGE gel --> transferred to a polymer sheet --> primary antibody (specific for POI) is added to sheet --> secondary antibody w/ enzyme (specific to primary ab) is added --> wash --> illuminate blot for identification + quantification of protein
52
primary vs secondary antibody in western blotting
- primary antibody: specific for protein of interest - secondary antibody: specific for primary antibody and attached to enzyme that has fluorescent tag
53
What is the purpose of western blotting?
- makes it possible to find a protein in complex mixture - identification and quantification of protein - useful to monitor protein purification and gene cloning
54
How does co-immunoprecipitation work?
incubate extract w/ monoclonal Ab specific for a protein --> add agarose beads coated with antibody-binding protein --> centrifuge to separate antibody-bound protein complex (left at bottom of tube) --> use other methods to identify bound protein (SDS-PAGE, western blot, mass spectro) | Antibody-binding protein recognizes Fc region ## Footnote Other proteins separate out during centrifugation = POI isolated with Ab and bead
55
Purpose of using fluorescent markers
- to visualize the cell and reveal cellular location - provides clues to protein function (from following movement of cell)
55
What does co-immunoprecipitation identify?
The binding partners of a protein by using a monoclonal antibody
56
Proteome
The entire set of proteins expressed and modified by a cell under a particular set of biochemical conditions - functional representation of genome | varies with cell type, environ conditions, and developmental stage
57
How does the proteome differ from the genome?
The proteome is not a fixed characteristic of the cell
58
X-ray crystallography
Reveals 3 dimensional structure in atomic detail
59
Electron density map
3d representation of where electrons are most densely localized + used to determine atom position in crystallized protein
60
How does x-ray crystallography work?
crystallize a protein --> expose crystal to source of x-rays --> detect diffracted x-rays --> interpret diffraction patterin into electron density map
61
How does x-ray crystallography work?
62
Mass spectrometry
Allows for the highly precise and sensitive detection of the mass of an analyte - measurement of atomic composition | can be done with no prior knowledge of identity
63
How does mass spectrometry work?
The analyte molecules get converted into gas-phase ions --> electrostatic potentials applied = **measure mass-to-charge ratio (m/z)**
64
3 essential components to mass spectrometry
- ion source (converts analyte to gas phase ion) - mass analyzer (distinguishes ions based on mass-to-charge ratios) - detector
65
time-of-flight (TOF) mass analyzer
Ions are accelerated through an elongated chamber under fixed electrostatic potential - ions w/ same net charge, small ion will move through faster --> mass of each ion can be determined by measuring time each ion passed through chamber
66
MALDI - matrix assisted laser desorption/ionization
Helps convert analyte into gas-phase ions - dry analyte out in presence of volatile, aromatic compound
67
MALDI-TOF
gas ions (generated from MALDI) pass directly into TOF analyzer which records mass-to-charge ratios - accurate way to measure protein mass
68
Peptide mass fingerprinting
Technique for individual protein identification - involves specific protein cleavage, chromatographic separation of resulting peptides, and mass spectrometry
69
Tandem mass spectrometry
Precursor ions can be broken into smaller chains --> new fragments called product ions can be passed through a second mass analyzer ## Footnote used for peptide sequencing
70
What does tandem mass spectrometry analyze?
Determines a proteins mass and amino acid sequence - breaks up parts of a large protein into fragments (product ions) ## Footnote fragmentation = breaks peptide bond
71
How does tandem mass spectrometry work?
It removes a terminal aa residue and compares the mass-to-charge ratio of the full peptide with the new fragment - diff. in mass-to-charge ratio can predict aa sequence

Chem 474 Biochemistry l (4 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions