5 - PROTEIN PURIFICATION AND CHARACTERIZATION TECHNIQUES

Question 1

a measurement of the amount of an enzyme recovered at each step of a purification experiment

Answer 1

Percent recovery

Question 2

the process of breaking cells open to release the organelles

Answer 2

Homogenization

Question 3

The simplest approach (homogenization) is grinding the tissue with a blender with a suitable buffer. The cells are broken open, releasing soluble proteins. This process also breaks many of the subcellular organelles (mitochondria, peroxisomes, and endoplasmic reticulum)

Question 4

A gentler technique is to use a Potter-Elvehjem homogenizer; The squeezing of the homogenate around the plunger breaks open cells, but it leaves many of the organelles intact.

Question 5

a thick-walled test tube through which a tight-fitting plunger is passed

Answer 5

Potter-Elvehjem

Question 6

involves using sound waves to break open the cells

Answer 6

Sonication

Question 7

a process in which ruptured cells are centrifuged several times increasing the force of gravity each time

Answer 7

Differential centrifugation

Question 8

Classic method of differential centrifugation:

1. First centrifugation (600 x g): At relatively low speed, the heavier components like unbroken cells and nuclei form a pellet, while smaller particles remain in the supernatant.
2. Second centrifugation (15,000 x g): The next step at a higher speed brings down mitochondria. The mitochondria form a pellet, while even smaller components remain in the supernatant.
3. Third centrifugation (100,000 x g): At this very high speed, you can pellet the microsomal fraction, which includes ribosomes and membrane fragments. Soluble proteins, if any, will stay in the supernatant.

Each step helps narrow down the sample, enriching it for the protein of interest if it’s soluble, while discarding heavier, less relevant cellular components.

Question 9

After the proteins are solubilized, they are often subjected to a crude purification based on solubility. Ammonium sulfate is the most common reagent to use at this step.

Question 10

a purification technique for proteins based on differential solubility in salt solutions

Answer 10

Salting out

Question 11

Proteins remains soluble because of their interactions with water.

Question 12

When ammonium sulfate is added to a protein solution, some of the water is taken away from the protein to make ion–dipole bonds with the salts. With less water available to hydrate the proteins, they begin to interact with each other through hydrophobic bonds.

Question 13

1. Initial salt addition: Ammonium sulfate is added to the solution, causing contaminating proteins to form a precipitate. These unwanted proteins are centrifuged out and discarded.
2. Further salt addition: More ammonium sulfate is added to increase the salt concentration. This causes another set of proteins, including the protein of interest, to precipitate. This precipitate is collected by centrifugation.
3. Salt concentration: The amount of ammonium sulfate is usually measured relative to a fully saturated solution. A common approach is to bring the solution to 40% saturation, remove the contaminants, and then increase it to 60-70% to get the proteins you want.

While not highly pure, this method is useful for initial protein separation before more refined purification steps.

Question 14

yt on differential centrifugation

Question 15

The word chromatography comes from the Greek chroma, “color,” and graphein, “to write”

Question 16

in chromatography, the substance that selectively retards the flows of the sample, effecting the separation

Answer 16

Stationary phase

Question 17

in chromatography, the portion of the system in which the mixture to be separated moves

flows over the stationary material and carries the sample to be separated along with it.

Answer 17

Mobile phase

Question 18

Some components interact relatively strongly with the stationary phase and are therefore carried along more slowly by the mobile phase than are those that interact less strongly. The differing mobilities of the components are the basis of the separation.

Question 19

a form of chromatography in which the stationary phase is packed in a column

Answer 19

Column chromatography

Question 20

The sample is a small volume of concentrated solution that is applied to the top of the column; the mobile phase, called the eluent, is passed through the column. The sample is diluted by the eluent, and the separation process also increases the volume occupied by the sample.

Question 21

yt on column chrom

Question 22

separates molecules on the basis of of size, making it a useful way to to sort proteins of varied molecular weights

the stationary phase consists of cross-linked gel particles

also called molecular-sieve chromatography

Answer 22

Size-exclusion chromatography/gel-filtration chromatography

Question 23

another name for gel-filtration chromatography which is a technique used to separate biomolecules based on size

Answer 23

Size-exclusion chromatography

Question 24

a type of column chromatography in which the molecules are separated according to size as they pass through the column

Answer 24

Gel-filtration chromatography

Question 25

carbohydrate polymer; often referred to by the trade names Sephadex and Sepharose

Answer 25

dextran or agarose

Question 26

a complex polysaccharide that is often used in column chromatography

Answer 26

dextran

Question 27

a complex polysaccharide used to make up resins for use in electrophoresis and in column chromatography

Answer 27

agarose

Question 28

sold under the trade name Bio-Gel

a form of electrophoresis in which a polyacrylamide gel serves as both a sieve and a supporting medium

Answer 28

Polyacrylamide

Question 29

Molecular-sieve chromatography advantages:

1. convenience as a way to separate molecules on the basis of size
2. it can be used to estimate the molecular weight by comparing the sample with a set of standards

Question 30

a powerful column separation procedure based on specific binding of molecules to a ligand

another form of column chromatography with a polymeric
material used as the stationary phase

has the advantage of producing very pure proteins

Answer 30

Affinity chromatography

Question 31

binds specifically to the desired protein

Answer 31

Ligand

Question 32

a method for separating substances on the basis of charge

interaction is less specific and is based on net charge

Answer 32

Ion-exchange chromatography

Question 33

a type of ion-exchange resin that has a net negative charge and bonds to positively charged molecules flowing through the column

Answer 33

Cation exchanger

Question 34

a type of ion-exchange resin that has a net positive charge and binds negatively charged molecules flowing through it

Answer 34

Anion exchanger

Question 35

Ion-exchange chromatography:

1. Equilibration: The column is prepared with a buffer (pH and salt conditions) and is filled with an exchange resin. This resin carries counterions, like Na⁺ or K⁺ for cation exchange, or Cl⁻ for anion exchange.
2. Protein Binding: A mixture of proteins is added. Proteins with the opposite charge of the resin will stick to it, replacing the counterions. Proteins with the same charge as the resin or no charge at all will flow through the column without sticking.
3. Elution: To release the bound proteins, the eluent is changed to either a buffer with a different pH or a higher salt concentration. This either neutralizes the charge of the proteins or outcompetes them for binding space, causing them to be washed off the column, leaving behind contaminants.

Question 36

a sophisticated chromatography technique that gives fast and clean purifications

Answer 36

High-performance liquid chromatography (HPLC)

Question 37

a form of high-performance liquid chromatography in which the stationary phase is nonpolar and the mobile phase is a polar liquid

Answer 37

Reverse phase HPLC

Question 38

a method for separating molecules on the basis of the ratio of charge to size

Answer 38

Electrophoresis

Question 39

most common support in electrophoresis is a polymer of agarose or acrylamide that is similar to those used for column chromatography.

Question 40

Electrophoresis:

A sample is applied to wells that are formed in the supporting medium. An electric current is passed through the medium at a controlled voltage to achieve the desired separation. After the proteins are separated on the gel, the gel is stained to
reveal the protein locations.

Question 41

are most often used to separate nucleic acids

Answer 41

Agarose-based gels

Question 42

is prepared and cast as a continuous cross-linked matrix, rather than being produced in the bead form employed in column chromatography.

Answer 42

Polyacrylamide gel

Question 43

In SDS-PAGE (a type of polyacrylamide-gel electrophoresis), proteins are treated with SDS, a detergent. SDS has a long structure (CH₃(CH₂)₁₀CH₂OSO₃⁻Na⁺) that binds to proteins, giving them a uniform negative charge and disrupting their normal shape by breaking noncovalent bonds. This means SDS denatures the proteins, causing them to unfold into a random coil, and ensures that proteins are separated by size alone (not by shape or charge) when they are applied to the gel for analysis.

Question 44

an electrophoretic technique that separates proteins on the basis of size

Answer 44

SDS-polyacrylamide-gel electrophoresis (SDS-PAGE)

Question 45

In SDS–PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), proteins are separated based on size. The gel (acrylamide) provides more resistance to larger proteins, so smaller proteins move faster through it. Since SDS masks the natural charge and shape of the proteins, their size is the only factor influencing movement. This method is similar to molecular-sieve chromatography and allows for estimating protein sizes by comparing them with known standards.

Without SDS, the gel is called a native gel, where proteins are kept in their natural form. In native gels, movement depends on size, shape, and charge, making it more complex to analyze.

Question 46

one without SDS or another compound that would denature the proteins being separated

Answer 46

Native gel

Question 47

a method for separating substances on the basis of their isoelectric points

different proteins have different titratable groups and thus have different isoelectric points

Answer 47

Isoelectric focusing

Question 48

Method in Isoelectric focusing:

• gel prepared has a pH gradient
• as proteins migrate through the gel, they encounter regions of different pH, so the charge on the protein changes.
• at the pI of a particular protein, it would no longer migrate, thus allowing separation from the other proteins

Question 49

2D gel electrophoresis (2D gel)

1. Isoelectric focusing: separation by charge
2. SDS-PAGE (run at 90 degrees to the first): separation by size

Question 50

Determining the primary structure of a protein:

1. establish which amino acids are present and in what proportions
2. the identities of the N-terminal and C-terminal amino acids in a protein sequence are determined.

3 & 4. the protein is cleaved into smaller fragments, and the amino acid sequence is determined.

Question 51

Breaking a protein down to its component amino acids is relatively easy: heat a solution of the protein in acid, usually 6 M HCl, at 100°C to 110°C for 12 to 36 hours to hydrolyze the peptide bonds

Question 52

a method for determining the amino acid sequence of peptides and proteins

Answer 52

Edman degradation

Question 53

Why are the proteins cleaved into small fragments for protein
sequencing?

Answer 53

The Edman degradation method, used to sequence proteins by identifying amino acids one at a time, becomes more challenging as the protein chain gets longer. Since most proteins are over 100 amino acids, scientists typically break the protein into smaller fragments (around 20 to 50 amino acids) to make the sequencing easier. This helps with the accuracy and efficiency of the process.

Question 54

a proteolytic enzyme specific for basic amino acid residues as the site of hydrolysis

cleaves peptide bonds preferentially at amino acids that have positively charged R groups (lysine and arginine)

The cleavage takes place in such a way that the amino acid with the charged side chain ends up at the C-terminal end of one of the peptides produced by the reaction

Answer 54

Trypsin

Question 55

a proteolytic enzyme that preferentially hydrolyzes amide bonds adjacent to aromatic amino acid residues ( tyrosine, tryptophan, and phenylalanine)

The aromatic amino acid ends up at the C-terminal ends of the peptides produced by the reaction

Answer 55

Chymotrypsin

Question 56

a reagent that cleaves proteins at internal methionine residues

The sulfur of the methionine reacts with the carbon of the cyanogen bromide to produce a homoserine lactone at the C-terminal end of the fragment

Answer 56

Cyanogen bromide

Question 57

When a protein is broken down by specific chemicals, it creates a mix of smaller pieces called peptides. These peptides are separated and analyzed using a technique called high-performance liquid chromatography. By using different chemicals on different samples of the same protein, you get different sets of peptides. Since these peptide sets overlap, you can piece together the complete protein sequence by determining the order of the peptides from the different sets.

Question 58

The actual sequencing of each peptide produced by specific cleavage of a protein is accomplished by repeated application of the Edman degradation.

Question 59

In the sequencing of a peptide, the Edman reagent, phenyl isothiocyanate, reacts with the peptide’s N-terminal residue.

Question 60

an automated instrument used in determining the amino acid sequence of a peptide or the nucleotide sequence of a nucleic acid

Answer 60

Sequencer

Question 61

practice on those apply your knowledge exercise

Question 62

To understand why proteins are cut into smaller pieces before sequencing, imagine trying to sequence a 100-amino-acid protein in one go. Although it seems simple, the process used, called Edman degradation, presents challenges.

In this method, a peptide reacts with phenylisothiocyanate (PITC), which attaches to one amino acid at a time, creating a derivative that we can analyze. Ideally, each peptide molecule would react with exactly one PITC molecule. However, in reality, it’s hard to get the quantities to match perfectly. For example, if we start with 100 peptide molecules but only 98 PITC molecules, we’ll get a mix of correctly processed and unprocessed amino acids. As the process repeats for the second amino acid, any leftover signals from previous steps can interfere, causing increasing confusion over time.

Therefore, proteins are cut into smaller pieces to make the process more manageable, allowing the sequence to be analyzed before the data gets too messy to interpret.

Question 63

a technique that separates molecular fragments according to their mass-to-charge ratio

Answer 63

Mass spectroscopy (MS)

Question 64

a form of mass spectrometry in which the sample is converted to fine droplets by spraying

Answer 64

Electrospray ionization (ESI-MS)

Question 65

a technique in which the output from one spectrometer is analyzed in a second spectrometer

Answer 65

Tandem mass spectrometry

Question 66

a technique that uses a laser to ionize the protein sample for MS

Answer 66

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS)

Question 67

enzyme-linked immunoabsorbent assay

Answer 67

ELISA

Question 68

an antibody that will react with the protein being studied or sought during an experiment

Answer 68

Primary antibody

Question 69

an antibody that will react with the primary antibody used in an ELISA or Western Blot experiment

Answer 69

Secondary antibody

Question 70

a technique where proteins are first separated using gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane for analysis and identification

Answer 70

Western blot

Question 71

The name, western blot, comes from a “tongue in
cheek” derivation of all blotting techniques.

Question 72

idk if i made a note already but make a separate docu on the pictures in the ebook + study it

Question 73

also called protein microarrays; small plates of a few cm on a side that can have tens of thousands of proteins implanted

Answer 73

Protein chips

Question 74

A protein chip, on the other hand, may have 30,000
separate samples stuck on a chip a few centimeters on a side. Fluorescence is the most common way to see the results.

Question 75

study of interactions among all the proteins of the cell

is the systematic analysis of an organism’s complete complement of proteins, its proteome, and it is
one of the fastest-growing fields.

Answer 75

Proteomics

Question 76

the total protein content of the cell

Answer 76

Proteome

Question 77

Proteomics is often subdivided into three basic types.

Answer 77

Structural, expression, and interaction

Question 78

offers a detailed analysis of the structure of the proteins being produced

Answer 78

Structural proteomics

Question 79

analyzes the expression of proteins, and frequently
considers their expression under different cellular conditions. It is a major contributor to our understanding of metabolism and disease.

Answer 79

Expression proteomics

Question 80

offers us the opportunity to look at how proteins interact with
other molecules.

Answer 80

Interaction proteomics

Question 81

How do the individual protein techniques combine to study
proteomics?

Answer 81

To put it simply, scientists use a combination of methods to figure out which proteins interact with each other inside cells. In this study, they created a “bait” protein, which they tagged so it could be easily tracked. The bait was mixed with cell components, and any proteins that stuck to it were captured using a special column. Afterward, they purified the protein complex, broke it down with trypsin (a protein-digesting enzyme), and used mass spectrometry to identify the proteins.

Question 82

Homogenization techniques:

Answer 82

grinding in a blender
grinding in a mortar (w/ liquid nitrogen)
using a Potter-Elvejhem homogenizer
Sonication
freezing and thawing
use of detergents (if the protein of interest is attached to a membrane)

Question 83

Differential centrifugation:

as the cell homogenate is subjected to increasing centrifugal force, different cell components end in the pellet.

Question 84

Size-exclusion chromatography:

When a sample is applied to the column, smaller
molecules, which are able to enter the pores, tend to be delayed in their progress down the column, unlike the larger molecules. As a result, the larger molecules are eluted first, followed later by the smaller ones, after escaping from
the pores

Question 85

a size of protein that is too large to fit inside the pores; all proteins that size or larger will elute first and simultaneously

Answer 85

Exclusion limit

Question 86

Affinity chrom:

other proteins in the sample not bound are eluted with buffer

bound protein is then eluted by adding high conc. of the ligand in soluble form, thus competing for biding of the protein with the stationary phase

protein binds to the ligand in the mobile phase and recovered from the column

protein ligand interaction can also be disrupted with a change in pH or ionic strength

Question 87

in a mixture of proteins, only one (designated P1) binds to a substance…

attached to the column matrix

Answer 87

Substrate

Question 88

Trends show a reduction in total protein and volume, with an increase in specific activity, while percent recovery typically decreases after each step

Question 89

The specific activity is a measure of the activity of the enzyme divided by the weight of protein in the sample. The higher the number, the more pure the sample

Question 90

Electrophoresis:

Macromolecules have differing mobilities based on their charge, shape, and size.

The anion binds strongly to proteins via nonspecific adsorption. The larger the protein, the more of the anion it adsorbs. SDS completely denatures proteins, breaking
all the noncovalent interactions that determine tertiary and quaternary structure. This means that multisubunit proteins can be analyzed as the component polypeptide chains. All the proteins in a sample have a negative charge as a result of adsorption of the anionic SO32. The proteins also have roughly the same shape, which is a random coil.