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Biochemistry > Chapter 3- Protein purification > Flashcards

Chapter 3- Protein purification Flashcards

1
Q

Assay

A

A type of test conducted by the individual that can be used to identify the protein of interest based on its unique function and properties. A positive test result indicates the presence of the protein in the mixture. For example, one property of reduced nicotinamide dinucleotide (NADH) is that it has the ability to absorb light of a specific wavelength (340 nm). Therefore we can monitor the activity of the enzyme indirectly by examining how much light is absorbed by the solution

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2
Q

In conducting an assay, which two questions must be answered?

A
  1. Is the protein present?

2. If present, what is the concentration of that protein?

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3
Q

Cell fractionation

A

A method of extracting the protein of interest from a sample of cells

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4
Q

Proteome

A

The entire set of proteins expressed and modified by a cell under a particular set of biochemical conditions

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5
Q

Homogenate

A

A mixture of all of the components of a cell- nuclei, organelles, etc.

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6
Q

Cell fractionation steps (2)

A
  1. The cells (in a test tube) are disrupted by some type of grinding process to create a homogenate
  2. The homogenate mixture is then allowed to undergo a series of differential centrifugation processes
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7
Q

Pellet

A

The bigger and heavier components move to the bottom of the test tube during centrifugation. As the force of centrifugation increases, new components are exposed. When the homogenate is centrifuged at low speed, a supernatant and pellet are created, and additional ones are created as the speed increases.

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8
Q

Supernatant

A

The lighter and smaller components remain at the top during centrifugation. As the force of centrifugation increases, new components are exposed. When the homogenate is centrifuged at low speed, a supernatant and pellet are created, and additional ones are created as the speed increases.

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9
Q

Differential centrifugation

A

Centrifugation separates materials (biological particles) using angular motion. Test tubes containing homogenates are accelerated to high rotational speeds. It is repeated several times to yield a series of pellets, which are enriched by different cellular materials. For example, one pellet will contain the nucleus, another will contain the mitochondria, and so on. It also produces a final supernatant, the soluble portion of the cytoplasm.

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10
Q

Salting out

A

The extent to which proteins dissolve in water depends on the amount of hydrophilic amino acids present in that particular protein. When you add salts (like NaCl) to a solution of protein dissolved in water, the solubility of the protein decreases. If you continue adding salt, the protein will become insoluble and will precipitate out of the solution. This is called salting out. Salting out takes advantage of the fact that the solubility of proteins varies with salt concentration.

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11
Q

When you add salt to a solution of protein dissolved in water, the solubility of the protein decreases. Why?

A

Most proteins require some salt to dissolve in water (salting in). When no salt is present in the solution, there are plenty of water molecules present that can interact with the charged and hydrophilic amino acids of the protein- this stabilizes the structure through hydrogen bonds. It is only when more and more salt is added that proteins become insoluble- the concentration of salt that makes a protein insoluble will vary from protein to protein.

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12
Q

With a high salt concentration, why will proteins not dissolve in water?

A

With a high salt concentration, the charged ions interact with water. This means that there are less solvent water molecules to interact with the protein. As a result, protein molecules begin to aggregate, which causes precipitation. The number of hydrogen bonds available to interact with hydrophilic amino acids will decrease, making the structure less stable- aggregation makes the structure more stable

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13
Q

What is salting out used for?

A

Salting out can be used to isolate a protein of interest from a mixture of different proteins based on the varying solubilities of proteins

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14
Q

How is salt removed from a protein solution after salting out?

A

Dialysis

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15
Q

Dialysis

A

The protein solution is placed in a cellophane bag with pores that are too small to allow the protein to diffuse through but big enough to allow the salt to diffuse through. Some salt will leave the bag to establish equilibrium- proteins will want to establish equilibrium as well, but they are too large to leave the bag

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16
Q

Dialysis takes advantage of which law?

A

The second law of thermodynamics- when given a chance to, energy will disperse to fill a greater region of space. Therefore over time, the majority of the small molecules (ions) will pass through the semipermeable membrane and out of the bag

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17
Q

What is gel filtration chromatography used for?

A

The separation of proteins based on size. Proteins are very diverse in terms of the length of their amino acid sequence.

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18
Q

What occurs during gel filtration chromatography?

A

A glass column is filled with porous beads. When a protein solution is passed over the beads, large proteins cannot enter the beads and exit the column first. Small proteins can enter the beads, therefore they have a longer path and exit the column last.

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19
Q

Gel filtration chromatography only works if

A

There is a relatively large size difference between the proteins

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20
Q

What is ion exchange chromatography used for?

A

Separation of proteins on the basis of charge. Depending on which amino acids a protein is composed of, the side chains will cause it to have a net positive, negative, or neutral charge in a specific pH.

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21
Q

What occurs during ion exchange chromatography?

A

A mixture of proteins is passed through the column of beads. Proteins with the same charge as the beads will exit the column quickly due to electrostatic repulsion. Proteins with the opposite charge will be attracted to the beads and will therefore barely travel at all

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22
Q

During ion exchange chromatography, how are oppositely charged proteins released?

A

A buffer is passed through the column- the proteins will be removed if the buffer has an increased salt concentration or if its pH is changed

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23
Q

Ion exchange chromatography is only helpful if

A

All proteins have a significantly different charge

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24
Q

Cation exchange chromatography

A

The beads have a negative charge- they are bound by positively charged proteins

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25
Q

Anion exchange chromatography

A

The beads have a positive charge- they are bound by negatively charged proteins

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26
Q

What is affinity chromatography used for?

A

Separating proteins on the basis of their affinity for certain chemicals or chemical groups. For example, antibody proteins bind to specific complementary antigens and DNA polymerase binds to specific sequences of nucleotides on the DNA for DNA replication

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27
Q

What occurs during affinity chromatography?

A

A column of beads is used- beads are made with a specific chemical attached. A protein mixture is passed through the column, and only proteins with affinity for the attached group will be retained.

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28
Q

In affinity chromatography, how are the bound proteins released?

A

A solution enriched with the chemical to which the protein is bound is passed through the column

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29
Q

Why is high performance liquid chromatography beneficial?

A

Very fine beads allow more interactions with the proteins and therefore improve purification, but flow rates through these columns are slow- HPLC improves the speed of this technique

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30
Q

How does high performance liquid chromatography work?

A

It uses very fine beads in metal columns and high pressure pumps to move the liquid through the column. Because of the increased number of interaction sites, the resolving power of HPLC is greater than normal columns.

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31
Q

Affinity chromatography is only useful if

A

We know what specific molecule that protein binds to and we know that the other proteins don’t bind to that specific molecule

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32
Q

In an electric field, proteins will migrate with a velocity directly proportional to (2)

A

Electrical field strength (E) and the charge on the protein (z)

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33
Q

In an electric field, proteins will migrate with a velocity inversely proportional to

A

The frictional coefficient (f)

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34
Q

Gel electrophoresis velocity equation

A

v= EZ/f

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35
Q

The frictional coefficient for gel electrophoresis is a function of (4)

A
  1. Protein mass
  2. Shape
  3. Radius (r)
  4. Density of the medium (n)
    f= 6pnr
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36
Q

Gel electrophoresis definition

A

When migration occurs in a gel

37
Q

What is gel electrophoresis used for?

A

Separating proteins on the basis of mass- the reagent used gives all proteins the same charge, meaning that charge is not a factor in movement of the protein

38
Q

What is the purpose of SDS in gel electrophoresis (SDS-PAGE)?

A

SDS breaks the non covalent interactions of protein, denaturing it. It also attaches onto the main amino acid side chain groups at a ratio of 1 attachment per 2 amino acids. SDS molecules have a negative charge, so they give all proteins a negative charge when they bind regardless of their native charge- makes the protein move in the electric field based on mass

39
Q

What type of gel is used in gel electrophoresis?

A

Polyacrylamide gel. Polyacrylamide is a polymer that does not react chemically with the proteins. It has many pores and channels as it folds that allow proteins to move through it. We don’t want the proteins to react and change their chemical nature

40
Q

How are proteins separated by SDS-PAGE visualized?

A

Proteins separated by SDS-PAGE are visualized by staining the gel with dyes such as Coomassie blue

41
Q

In gel electrophoresis, why do large proteins move more slowly?

A

Larger proteins move more slowly because they experience greater friction than smaller proteins. Therefore, larger proteins will be higher up along the slab than smaller proteins. Larger proteins are also bound by more SDS molecules since they have more amino acids. This is how gel electrophoresis is able to indicate mass.

42
Q

Isoelectric point (pI)

A

SInce proteins consist of different combinations of these ionizable amino acids, they will have different net charges at the physiological pH of around 7. It also means that every protein will have a unique pH value at which the overall net charge will be zero (all charges will cancel out). In fact, this pH value at which the protein has a net charge of zero is called the isoelectric point (pI). This is a property of the protein, since they all have unique amino acids

43
Q

What occurs during isoelectric focusing?

A

Each protein will move due to the presence of an electric field. When they reach the isoelectric point, their net charge of zero will cause them to stop. The proteins have a net charge, so they will move due to the electric field- a protein with a negative charge will move towards the positive end of the electric field and vice versa
They will move until they reach the pH value where their overall charge is 0. At this point, they will not move because they no longer have a charge and are not attracting or repelling the charge of the electric field.

44
Q

How is isoelectric focusing set up?

A

A gel with a pH gradient is set up. The two ends are connected to a voltage source and the proteins are placed into the gel. Left side of gel is acidic, right side is basic

45
Q

Which amino acids are uncharged when protonated? (2)

A

Asp and Glu. They have carboxyl groups

46
Q

Which amino acids are uncharged when deprotonated? (3)

A

Lys, Arg, and His, which have various types of N-containing groups

47
Q

Two dimensional gel electrophoresis definition

A

Isoelectric focusing can be combined with SDS-polyacrylamide gel electrophoresis to create a more effective means of purifying a mixture of proteins.

48
Q

First step of two dimensional gel electrophoresis

A

A mixture of proteins is first exposed to isoelectric focusing. This separates the proteins based on their isoelectric points (pH values at which the net charge on the protein is zero). If two or more proteins have the same pI value, they will be found in the same band. Like usual, proteins stop moving when their net charge is zero. Many proteins can exist in the same band, in this case (if they have the same pI value). This is the horizontal component.

49
Q

Second step of two dimensional electrophoresis

A

The horizontal gel lane that contains the proteins is then placed into the SDS-PAGE apparatus, separates the proteins based on size. Now, proteins with the same pI value are located at the same value on the x axis, but are separated vertically by size. The proteins now begin to move in a perpendicular direction downward. This will separate proteins with identical pI values but different masses.

50
Q

Two-dimensional electrophoresis can be used to

A

Detect differences in protein expression under different physiological circumstances.

51
Q

How can we determine if our method of protein purification is working? (3)

A
  1. Total protein (mg)- this tells us how much protein is present in our mixture
  2. Enzyme activity
  3. Specific activity- this is the ratio of enzyme activity to total protein. It tells us how pure our sample is
52
Q

Enzyme activity

A

This describes the ability of the enzyme to promote a particular reaction within the sample. It tells us the millimoles of product produced per minute (unit) in the mixture

53
Q

Specific activity

A

Tells us how pure the protein sample is. It’s the ratio of enzyme activity to total protein concentration. Specific activity should
increase with each step of the purification procedure since total protein is being removed while desired enzyme is being retained. SDS-PAGE allows a visual evaluation of the purification scheme.

54
Q

Zonal (gradient) centrifugation

A

A density gradient is formed in a centrifuge tube and a mixture
of proteins in solution is placed on top of the gradient. After the centrifugation is complete, a small hole is made in the bottom of the tube and portions of the gradient are collected. These portions are analyzed for protein concentration, enzyme activity, or other biochemical characteristics

55
Q

Uses of recombinant DNA technology (3)

A
  1. Large quantities of proteins can be obtained.
  2. Proteins can be modified with affinity tags that allow purification of the protein or visualization of the protein in the cell.
  3. Proteins with modified primary structure can be generated.
56
Q

Antibodies

A

Proteins created by the organism that serve a protective function. In the presence of a foreign pathogen, the immune cells begin producing these antibodies. Antibodies are highly specific proteins that have a strong affinity for antigens.

57
Q

Antigens

A

A molecule recognized by an antibody. Any pathogenic molecule, such as a sugar, a nucleic acid, or a protein, can serve as an antigen. Tiny peptide segments can also act as antigens.

58
Q

Which structures on an antigen are recognized by antibodies?

A

The antibody recognizes a particular structural feature on the antigen called the antigenic determinant or epitope

59
Q

Monoclonal antibodies

A

Any antibody-producing cell synthesizes antibodies that recognize only one epitope. Each antibody-producing cell thus synthesizes a monoclonal antibody. Monoclonal antibodies are formed from a single type of plasma cell and always bind to a single epitope.

60
Q

Polyclonal antibodies

A

Any antigen may have multiple epitopes. The antibodies produced to the antigen by different types of plasma cells are said to be polyclonal.

61
Q

How can cell lines of monoclonal antibodies be generated?

A

Immortal cell lines producing monoclonal antibodies can be generated by fusing normal antibody-producing cells with cells from a type of cancer called multiple myeloma. A monoclonal cell line is isolated by screening for the antibody of interest.

62
Q

ELISA definition

A

Antibodies can be used as a reagent to quantify the amount of a protein or other antigen. Enzyme-linked immunosorbent assay (ELISA) quantifies the amount of protein present because the antibody is linked to an enzyme that produces a readily identified
colored product

63
Q

Indirect ELISA is used for

A

This method is used to test for the presence of a specific antibody- an individual with HIV will produce antibodies against the HIV antigen- we can use indirect ELISA to test for these antibodies.

64
Q

Indirect ELISA process

A

The surface of the well is coated with a specific antigen. Mixtures of antibodies extracted from the blood and are added to the well. If the antibody of interest is present, it will bind to the antigen. Enzyme-linked antibodies that can bind to the antibody of interest are added into the well. If the antibody of interest is bound to the antigen, the enzyme linked antibody will bind to the antibody-antigen complex. Unbound antibodies are removed by washing. The substrate specific to the enzyme is then added. If the enzyme is present, the substrate will react and cause a color change. This signifies the presence of the antibody of interest

65
Q

Sandwich ELISA is used for

A

Generally used to detect the presence of an antigen rather than the antibody

66
Q

Sandwich ELISA process

A

The antibody specific to the antigen is attached to the bottom of the well. Some sample containing the antigen, such as blood or urine, is placed into the well. The antigen then binds to the antibody. Now, an enzyme-linked monoclonal antibody is added. It binds to the antigen attached to the adsorbed antibody. At this point, we have 2 antibodies with the antigen in between (sandwich)- if antigen isn’t present, the enzyme linked antibody has nothing to bind to. The well is then washed to remove anything unbound. The substrate is added to react with the enzyme, producing the colored product.

67
Q

What is western blotting used for?

A

Western blotting is a method that utilizes antibodies to detect the presence of a protein in some sample (cell fluid, blood plasma, etc). The point is to form a specific antibody that can bind onto that protein that we want to locate. We can centrifuge the cell to get the protein, then use SDS gel electrophoresis to separate proteins based on size

68
Q

Western blotting process

A

Proteins are separated in an SDS-PAGE gel, transferred to a polymer, and then stained with an antibody specific for the protein, called the primary antibody. At this stage, the protein is still not readily detectable. A second antibody, the secondary antibody, specific for the first antibody is then added. The secondary antibody is attached to an enzyme that generates a chemiluminescent product or contains a fluorescently labeled tag, thereby allowing the detection of the primary antibody and revealing the location of the protein of interest.

69
Q

What is the purpose of co-immunoprecipitation?

A

When a monoclonal antibody against a specific protein is available, it is also possible to determine the binding partners of that protein under cellular conditions

70
Q

Co-immunoprecipitation process

A

A cell extract is incubated with the antibody; then agarose beads coated with an antibody-binding protein (e.g., Protein A) are added to the mixture. The antibody-bound protein complex can be separated from all other cellular components. The identity of other bound protein partners can then be probed by other methods.

71
Q

What is mass spectrometry used for?

A

Mass spectrometry allows the highly accurate and sensitive detection of the mass of the molecule of interest, or analyte. This includes peptides and proteins. Matrix-assisted laser desorption/ionization (MALDI) is often combined with a time-of-flight (TOF) detector.

72
Q

Mass spectrometry components

A

Mass spectrometers consist of three components: an ion

source, a mass analyzer, and a detector

73
Q

Mass spectrometers function

A

Mass spectrometers convert the analyte into gas-phase

ions. The mass-to-charge ratio (m/z) can be determined

74
Q

Edman degradation

A

The amino acid sequence of a protein can be determined by
Edman degradation. The protein is exposed to phenyl
isothiocyanate (PTH), which reacts with the N-terminal amino acid to form a PTH derivative. The PTH–amino acid can be released without cleaving the remainder of the protein, and the degradation is subsequently repeated. High-performance liquid chromatography is used to identify the amino acids. This method has mostly been replaced by mass spectrometry.

75
Q

Why isn’t it possible to sequence large proteins?

A

Because the reactions of the Edman degradation and mass spectrometry procedure are not 100% effective, it is not possible to sequence polypeptides longer than 50 amino acids. To sequence the entire protein, the protein is chemically or enzymatically cleaved to yield peptides of fewer than 50 amino acids. The peptides are then ordered by performing a different cleavage procedure to generate overlap peptides.

76
Q

How are disulfide bonds determined?

A

Extra steps are required for the determination of disulfide bonds. Disulfide bonds are cleaved by the addition of a reducing agent. Disulfide-bond reformation is prevented by the alkylation of the cysteine residues.

77
Q

How are genomic and proteomic methods complementary?

A

Genomic techniques that reveal the DNA sequence of the gene encoding a protein also allow determination of the amino acid sequence.

78
Q

What information can the amino acid sequence of a protein provide? (6)

A
  1. Amino acid sequences of proteins can be compared to identify
    similarities.
  2. Comparison of the sequence of the same protein from different
    species yields evolutionary information.
  3. Amino acid sequence searches can reveal the presence of internal repeats.
  4. Sequencing information can identify signals that determine the
    location of the protein or processing signals.
  5. Sequence information can be used to generate antibodies for the protein.
  6. Amino acid sequence can be used to generate DNA probes
    specific for the gene encoding the protein.
79
Q

Purposes for synthesizing peptides (4)

A
  1. As antigens to produce antibodies
  2. To isolate receptors. Example: synthetic peptides containing formylmethionine (fMet) have been used to identify receptors on white blood cells.
  3. As drugs
  4. To understand protein folding
80
Q

How are unwanted reactions prevented in protein synthesis?

A

Some groups must be blocked and others must be activated. Synthetic peptides are synthesized in a stepwise fashion with the carboxyl terminus of the growing chain attached to an inert matrix. t-Boc blocks the amino group of the incoming amino acid, and DCC facilitates peptide bond formation.

81
Q

X-ray crystallography purpose

A

Reveals three-dimensional structure in atomic detail- crystals of proteins are irradiated with x-rays

82
Q

How does X-ray crystallography work? (3)

A
  1. Electrons of the atoms scatter x-rays.
  2. The scattered waves recombine.
  3. The way in which the scattered x-rays recombine reveals the
    atomic arrangement.
83
Q

How does NMR work?

A

NMR is based on the fact that certain atomic nuclei are intrinsically magnetic and can exist in two spin states when an external magnetic field is applied. The nuclei of the sample absorb electromagnetic radiation at different frequencies termed chemical shifts. The chemical shifts depend on the environment of the nuclei,
and the environment depends on protein structure

84
Q

One-dimensional NMR

A

Reveals changes to a particular chemical group under different conditions

85
Q

Two-dimensional NMR (NOESY)

A

Displays groups that are in close proximity.

86
Q

In NMR, a chemical shift line represents

A

The transition between the alpha and beta spin states

87
Q

Nuclear Overhauser Effect (NOE)

A

Identifies pairs of protons that are in close proximity based on proton chemical shift

88
Q

What is cryo-electron microscopy used for?

A

Many 2D projections are detected, each capturing a molecule
in a different orientation. A computer uses these projections
to build a three-dimensional representation of the protein.

89
Q

Cryo-electron microscopy process

A

To perform cryo-EM, a thin layer of the protein solution is prepared in a fine grid, then frozen very quickly. This traps the molecules in an ensemble of orientations. The sample is then placed in a transmission electron microscope under vacuum conditions and exposed to an incident electron beam. Each protein interacts with the beam to produce a two-dimensional projection on the detector

Biochemistry (13 decks)

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