5 - PROTEIN PURIFICATION AND CHARACTERIZATION TECHNIQUES Flashcards
a measurement of the amount of an enzyme recovered at each step of a purification experiment
Percent recovery
the process of breaking cells open to release the organelles
Homogenization
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)
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.
a thick-walled test tube through which a tight-fitting plunger is passed
Potter-Elvehjem
involves using sound waves to break open the cells
Sonication
a process in which ruptured cells are centrifuged several times increasing the force of gravity each time
Differential centrifugation
Classic method of differential centrifugation:
- 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.
- 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.
- 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.
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.
a purification technique for proteins based on differential solubility in salt solutions
Salting out
Proteins remains soluble because of their interactions with water.
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.
- 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.
- 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.
- 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.
yt on differential centrifugation
The word chromatography comes from the Greek chroma, “color,” and graphein, “to write”
in chromatography, the substance that selectively retards the flows of the sample, effecting the separation
Stationary phase
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.
Mobile phase
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.
a form of chromatography in which the stationary phase is packed in a column
Column chromatography
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.
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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
Size-exclusion chromatography/gel-filtration chromatography
another name for gel-filtration chromatography which is a technique used to separate biomolecules based on size
Size-exclusion chromatography
a type of column chromatography in which the molecules are separated according to size as they pass through the column
Gel-filtration chromatography
carbohydrate polymer; often referred to by the trade names Sephadex and Sepharose
dextran or agarose
a complex polysaccharide that is often used in column chromatography
dextran
a complex polysaccharide used to make up resins for use in electrophoresis and in column chromatography
agarose
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
Polyacrylamide
Molecular-sieve chromatography advantages:
- convenience as a way to separate molecules on the basis of size
- it can be used to estimate the molecular weight by comparing the sample with a set of standards
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
Affinity chromatography
binds specifically to the desired protein
Ligand
a method for separating substances on the basis of charge
interaction is less specific and is based on net charge
Ion-exchange chromatography
a type of ion-exchange resin that has a net negative charge and bonds to positively charged molecules flowing through the column
Cation exchanger
a type of ion-exchange resin that has a net positive charge and binds negatively charged molecules flowing through it
Anion exchanger
Ion-exchange chromatography:
- 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.
- 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.
- 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.
a sophisticated chromatography technique that gives fast and clean purifications
High-performance liquid chromatography (HPLC)
a form of high-performance liquid chromatography in which the stationary phase is nonpolar and the mobile phase is a polar liquid
Reverse phase HPLC
a method for separating molecules on the basis of the ratio of charge to size
Electrophoresis
most common support in electrophoresis is a polymer of agarose or acrylamide that is similar to those used for column chromatography.
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.
are most often used to separate nucleic acids
Agarose-based gels
is prepared and cast as a continuous cross-linked matrix, rather than being produced in the bead form employed in column chromatography.
Polyacrylamide gel
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.
an electrophoretic technique that separates proteins on the basis of size
SDS-polyacrylamide-gel electrophoresis (SDS-PAGE)
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.
one without SDS or another compound that would denature the proteins being separated
Native gel
a method for separating substances on the basis of their isoelectric points
different proteins have different titratable groups and thus have different isoelectric points
Isoelectric focusing
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
2D gel electrophoresis (2D gel)
- Isoelectric focusing: separation by charge
- SDS-PAGE (run at 90 degrees to the first): separation by size
Determining the primary structure of a protein:
- establish which amino acids are present and in what proportions
- 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.
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
a method for determining the amino acid sequence of peptides and proteins
Edman degradation
Why are the proteins cleaved into small fragments for protein
sequencing?
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.
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
Trypsin
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
Chymotrypsin
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
Cyanogen bromide
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.
The actual sequencing of each peptide produced by specific cleavage of a protein is accomplished by repeated application of the Edman degradation.
In the sequencing of a peptide, the Edman reagent, phenyl isothiocyanate, reacts with the peptide’s N-terminal residue.
an automated instrument used in determining the amino acid sequence of a peptide or the nucleotide sequence of a nucleic acid
Sequencer
practice on those apply your knowledge exercise
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.
a technique that separates molecular fragments according to their mass-to-charge ratio
Mass spectroscopy (MS)
a form of mass spectrometry in which the sample is converted to fine droplets by spraying
Electrospray ionization (ESI-MS)
a technique in which the output from one spectrometer is analyzed in a second spectrometer
Tandem mass spectrometry
a technique that uses a laser to ionize the protein sample for MS
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS)
enzyme-linked immunoabsorbent assay
ELISA
an antibody that will react with the protein being studied or sought during an experiment
Primary antibody
an antibody that will react with the primary antibody used in an ELISA or Western Blot experiment
Secondary antibody
a technique where proteins are first separated using gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane for analysis and identification
Western blot
The name, western blot, comes from a “tongue in
cheek” derivation of all blotting techniques.
idk if i made a note already but make a separate docu on the pictures in the ebook + study it
also called protein microarrays; small plates of a few cm on a side that can have tens of thousands of proteins implanted
Protein chips
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.
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.
Proteomics
the total protein content of the cell
Proteome
Proteomics is often subdivided into three basic types.
Structural, expression, and interaction
offers a detailed analysis of the structure of the proteins being produced
Structural proteomics
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.
Expression proteomics
offers us the opportunity to look at how proteins interact with
other molecules.
Interaction proteomics
How do the individual protein techniques combine to study
proteomics?
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.
Homogenization techniques:
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)
Differential centrifugation:
as the cell homogenate is subjected to increasing centrifugal force, different cell components end in the pellet.
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
a size of protein that is too large to fit inside the pores; all proteins that size or larger will elute first and simultaneously
Exclusion limit
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
in a mixture of proteins, only one (designated P1) binds to a substance…
attached to the column matrix
Substrate
Trends show a reduction in total protein and volume, with an increase in specific activity, while percent recovery typically decreases after each step
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
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.