Protein Lec UNIT 3

What are the 6 purification steps

Sample Selection
Homogenization
Crude fractionation (Centrifuge/Salting out)
High resolution separation (SDS-Page/Chromatography)
Quantitative Evaluation (Enzymatic Assay)
Sequencing primary polypeptide chain

Fix the error. In sample selection the chosen source must be abundant in the target molecules

No error

Why do we homogenize? (4)

Release target molecules
Maximize extraction yield
Sample uniformity
Produce a homogenate

____ is a thick extract containing soluble cytosolic components

Homogenate

What are 3 types of disruption mechanism

Physical
Chemical
Enzymatic

What are 5 .types of physical disruption method

Sonication
Mild Detergents
High-pressure extrusion
Mechanical shearing
Hypotonic osmotic shock

Factors affecting biomolecule stability (6)

pH
Temperature
Inhibitor
Oxidation/Reduction
Osmotic concentration
Heavy metal concentration

We use _____ to maintain stable _____ to prevent changes in side-chain ionization

buffer solutions, pH

We keep procedures _____ at because heat disrupts weak non-covalent interaction causing thermal degradation

cold

Fix the error. A good assay targets multiple molecules

targets 1 molecule

Fix the error. A good assay should only be able to detect high amounts

trace amounts

An example of assay strategy is to measure the rate of ______ or ______ over time via spectrophotometry

product formation, substrate disappearance

Fix the error. In coupled enzymatic reaction, the primary reaction yields a detectable result

undetectable result

Why do we use a second enzyme after the primary reaction in couple enzymatic reaction

To convert the invisible/undetectable product of the first reaction to something measurable

What are 4 separation based on solubility

Isoelectric precipitation
Salting-in/out
Organic solvent
Metal chelators/detergentt

Why do electrostatic repulsion vanishes between protein molcules during isoelectric precipitation

Because the protein is in a zwitterion state

Why is the result of the protein entering a zwitterionic causes it to precipitate

hydrophobic patches opens up and interacts aggregating

What is the shape of solubility curve

U

Salting in _____ solubility at _____ ionic strength

increases, low

Salting out increases _____ at _____ ionic strength

precipitation, high

What is salt used for salting-in/out

NaCl & Ammonium sulfate (NH₄)₂SO₄

Fix the error. At high dielectric constant, there is more organic solvents

Water

Fix the error. We add inorganic solvents to decrease the dielectric constant to decrease aggregation

organic solvents decreases (D) increasing aggregation

Fix the error. At high D the sample is likely to aggregate

Low D

What are the two parts produced after centrifugation

Supernatant
Pellet

What are 3 forces acting on sedimenting particle

Centrifugal
Buoyant
Frictional

Fix the error. Heavier components exhibit lower S value

Lighter components

Fix the error. Higher S value allows for faster sedimentation at lower speeds

No error

What are 3 types of centrifugal rotor

Fixed angle
Swinging bucket
Vertical

_____ has the fastest run time of all centrifugal rotor

Vertical rotors

What are 3 advantages of fixed-angle rotors

Short pathlength
Rapid pelleting
Higher RPM

What are 3 advantages of swinging bucket rotor

Long pathlength
Superior resolution
Easier supernatant extraction

What are 2 advantages of vertical rotor

Shortest pathlength
Fastest runtime

What are 4 types of centrifuge classification and their matching RPM

Lowspeed (1,000-6,000)
Mediumspeed (12,000-15,000)
Highspeed (1,000-25,000)
Ultracentrifuge (20,000-80,000)

_____ has a vacuum chamber, refrigerated; isolates ribosomes, viruses, macromolecules, and allows analytical measurement of molecular mass.

Ultracentrifuge

_____ is refrigerated (0–4°C); used for cell organelles, microsomal mfraction, bigger speed range

High-speed

_____ is for benchtop pelleting of nucleic acids/proteins, also called “microfuge”

Medium-speed

_____ is unrefrigerated clinical or desk-top; used for whole cells and red blood cells.

Low-speed

What are 2 types of centrifugation techniques

Differential centrifugation
Density gradient centrifugation

Describe differential centrifugation

Separation of components from big to small at an increasing speed

What is dialysis

Passive diffusion of small solutes across semi-permeable membrane down concentration gradient

_____ uses osmotic principle to passively diffuse small solute particles across a semipermeable membrane from high to low concentration

Dialysis

______ is a centrifugation where particles migrate until their buoyant density matches surrounding solution density

Isopycnic (equilibrium) Centrifugation

______ is a centrifugation where bands separate based on size and shape of S value as they travel down.

Rate-zonal (s-value) centrifugation

_____ is used for desalting solutions, exchanging buffer components, and removing unbound cofactors or inhibitors.

Dialysis

_____ is used for rapidly concentrating dilute protein samples or desalting.

Ultrafiltration

_____ elute first in a GF/SE chromatography

Large

In a GF/SE chroma the stationary phase is made up of _____

porous beads

What are 3 gels used in GF/SE chroma

Dextran (Sephadex)
Agarose (Sepharose)
Polyacrylamide (Bio-gel)

What is the order of elution in a GF/SE chroma?

Elute in decreasing size

Why does small particles elute last in a GF/SE chroma

Because they enter porous beads traversing a much larger effective path length

What are 2 separation based on charge

Paper electrophoresis
Isoelectric focusing

Characteristics of gel filtration media (4)

Chemically inert
Uncharged
Uniform pore size
High mechanical rigidity

Dextran is produced by _____

Leuconostoc mesenteroides

Describe agarose

Linear polymer of alternating D-galactose and 3,6-anhydro-L-galactose from red algae

In GF/SE Chroma, what is Ve

Volume solvent required to elute solute from column after it interacts with gel

in GF/SE chroma, what is Vo

Volume of solvent space surrounding beads

In GF/SE chroma, what is Vx

Volume occupied by gel beads

In GF/SE chroma, what is Vt

Total bed volume of column

In Ve=Vo what happens

It elutes fastest because it is too big

In Vo<Ve<Vt what happens

Elutes 2nd as it is within pore range

In Ve=Vt what happens

Elutes 3rd it is tiny

In Ve>Vt what happens

Elutes last because it is sticky/interacting with bead

Fix the error. When choosing a gel it is always better to choose a fractionation range smaller than target

Choose a fractionation range where your target is in the middle

What happens if you use a too small fractionation range

Your sample will not be the only one detected, it will detect other much bigger components

Gel-electrophoresis is a type of _____ cell

Electrolytic

In a gel-electrophoresis the cathode is? (3)

Region of high pH concentration
Reduction occurs
Attracts cation

In a gel-electrophoresis the anode is? (3)

Region of low pH concentration
Oxidation occurs
Attracts anion

In gel-electrophoresis the travel is from _____ to _____

cathode, anode

In electrophoresis - Anode : Cathode , _____ electrode : _____ electrode

Positive, Negative

_____ is the movement of charged molecules through a gel/support matrix driven by an electric field.

Electrophoresis

PAGE stands for?

Polyacrylamide Gel-electrophoresis

_____ is a protein analysis technique that drives proteins along a pH gradient until they stop at their pI

Isoelectric focusing

What is the separation sensitivity of isoelectric focusing?

0.01 pH

The pH gradient in isoelectric focusing is established by _____

Ampholytes

_____ is a protein analysis technique where proteins remain folded in their native state. Migration depends simultaneously on charge, size, and shape.

Native-PAGE

SDS stands for?

Sodium Dodecyl Sulfate

_____ is an ampipathic detergent that denatures secondary tertiary and quarternary protein structure by binding to the hydrophobic polypeptide bacbone

Sodium Dodecyl Sulfate (SDS)

Why are proteins denatured in SDS-page

To ensure that proteins are separated based on their size rather than their charge

Applications of SDS-Page (2)

Purity assessment
Determine molecular weight

In SDS-Page _____ or _____ is added to cleave covalent disulfide bonds

DTT, 2-mercaptoethanol

In electrophoresis the _____ is the starting point

Cathode

What are two discontinuous buffer system in SDS-Page

Stacking gel
Resolving (running) gel

Characteristics of stacking gel? (3)

Upper gel
Low PA concentration (4%)
Low pH (6.8)

Characteristics of resolve (running) gel (3)

Lower gel
High PA concentration (8-15%)
High pH (8.8)

What are electrophoresis visualization methods (3)

Coomassie brilliant blue
Silver staining / Fluorescamine
Western (Immuno) blot

_____ is a visualization method that detects microgram quantities proteins

Coomassie brilliant blue

_____ is a visualization methods that is highly sensitive (nanogram scale)

Silver staining / Fluorescamine

Fix the error. Agarose forms much smaller pore sizes that polyacrylamide

Agarose forms bigger pore sizes

Describe the 2 dimensions in 2D gel electrophoresis

1st dimension: uses isoelectric focusing in a tube gel
2nd dimension: tube gel placed on SDS-Page slab gel

2D gel electrophoresis first separates strictly by _____ then separates it strictly by _____

isoelectric point, molecular weight

______ separates biomolecules based on reversible electrostatic interactions between charged protein side chains and charged functional groups on an insoluble matrix

Ion Exchange chromatography

What are 2 exchanger types

Anion exchanger
Cation exchanger

Anion exchangers contains _____ matrix and bind to _____

positively charged, anions

Cation exchangers contains _____ matrix and bind to _____

negatively charged, cations

_____ is added to sample buffer to indicate progress of electrophoresis run

Tracking dye

_____ is used if sample is radioactive

Autoradiograph

What are two separations based on polarity and specific binding

Affinity chromatography
Reverse-phase chromatography

Describe the phases in reverse phase chromatography

Stationary: Non polar
Mobile: Polar