Methods to Study Proteins

in life

in vivo

in glass

in vitro

a protein is studied inside the intact cell in its native context (with all other proteins and cellular components present)

in vivo

an individual isolated protein (or set of proteins) is studied in a test tube under controlled conditions

in vitro

Cell

Proteins Must Be Removed from the what to Be Purified

lysate

Cells are disrupted to form a homogenate or what, which is a mixture of all the components of the cell

differential centrifugation

The lysate is centrifuged at low speed to yield a pellet consisting of nuclei, and a supernatant. This supernatant is then centrifuged at a higher centrifugal force to yield another pellet and supernatant

cytoplasm

This process, called differential centrifugation, is repeated several more times to yield a series of pellets enriched in various cellular materials and a final supernatant that contains
proteins present in the what of the intact cells

soluble

This process, called differential centrifugation, is
repeated several more times to yield a series of pellets enriched
in various cellular materials and a final supernatant that contains
proteins present in the cytoplasm of the intact cells

• these are ‘what’ proteins (the majority)

membrane associated

This process, called differential centrifugation, is
repeated several more times to yield a series of pellets enriched
in various cellular materials and a final supernatant that contains
proteins present in the cytoplasm of the intact cells

• there are also ‘what’ proteins

assay

protein purification requires a test or what, so you can know that you have your protein and determine how much of the protein of interest is present

activity

An assay for the enzyme lactate dehydrogenase relies on the fact that a
product of the reaction, NADH, can be detected with a spectrophotometer
- this is an assay based on protein ‘what’

rate

enzyme activity is expressed as a what = amount of product formed/time

investigator

defines a ‘unit’ of activity

specific activity

protein purifications are monitored in part by determining the what of the protein as it is purified

specific activity

amount of enzyme activity/amount of total protein

specific activity should what with each step of the purification procedure

increase

Sequential Purification Steps does what to Sample Size

Decrease

Binding

Proteins Can Be Purified Based on Solubility, Size, Charge, and what Affinity

solubility

‘salting out’ takes advantage of the fact that the what of proteins varies with the salt concentration

Most proteins require some salt to dissolve in water, a process called salting what

in

Most proteins will denature and precipitate at different salt concentrations, a process called ‘salting what’

out

now the protein you want to study has denatured and you can’t
study it, because it is no longer folded into its native shape and therefore
will be what

inactive

dialysis

The salt can be removed from a protein solution by what

renature

As the salt dialyses out of the bag the proteins will refold or ‘what’ and regain activity

purification factor

new specific activity/previous specific activity

chromatography

using a column to separate a mixture of proteins based on differences in their size, charge or binding properties

aqueous

an what solution containing a mixture of proteins is run through a column containing some solid matrix that separates proteins based on their size, charge or affinity

fractions

Column Chromatography: as the liquid runs through the column, ‘what’ of a fixed volume are collected at the bottom

activity

protein activity in each fraction is assayed and only the fractions with what are kept

charge

Ion-Exchange Chromatography separates based on a protein’s electric what

column

What matrix is a compound with a negative or positive charge

proteins with the same charge as the column matrix move what through the column

quickly

proteins with the opposite charge are attracted to the column and move more what through it

slowly

Proteins absorb light at what wavelength nm due to aromatic amino acids

280

spectrophotometer

A small amount of each fraction is measured in a what at l = 280nm to determine how much protein is in each fraction

gel

Size-Exclusion Chromatography also called what filtration chromatography

size

Size-Exclusion Chromatography separates based on protein what

column matrix is a compound with holes of a defined size (think a wiffle ball)
• which proteins do not go inside the beads and run through the column faster

large

column matrix is a compound with holes of a defined size (think a wiffle ball)
• which proteins go through the pores and ‘see’ the inside of the beads, and take a longer time to run through the column

small

bind

Affinity Chromatography separates proteins based on their affinity (ability to what) some other protein or compound, which is present on the column matrix

salt

proteins that bind to the affinity matrix will stick to the column, all others will wash through, bound proteins are then eluted by washing the column with a high concentration of what or the free ligand

High-Performance Liquid Chromatography (HPLC) uses what-pressure pumps to push proteins through the column

high

resolution

High-Performance Liquid Chromatography (HPLC) greatly improves what

Isoelectric focusing

allows separation of proteins in a gel based on their relative amounts of acidic and basic amino acids

Is Isoelectric Focusing a type of chromatography

No

point

If a mixture of proteins is placed in a gel with a pH gradient and an electrical field is applied, proteins will migrate until they reach their isoelectric what

net

If a mixture of proteins is placed in a gel with a pH gradient and an electrical field is applied, proteins will migrate until they reach their isoelectric point (pI), the pH at which they have no what charge, where they stop migrating

total purification

final specific activity/starting specific activity

electrophoresis

visualize and characterize purified proteins

Separated

Proteins Can Be what and Visualized by Protein Gel Electrophoresis

polyacrylamide

Polyacrylamide Gel Electrophoresis (PAGE) for Protein Analysis
• use cross-linked polymer of what to make a vertical gel

buffer

Polyacrylamide Gel Electrophoresis (PAGE) for Protein Analysis

what is present at top and bottom

Polyacrylamide Gel Electrophoresis (PAGE) for Protein Analysis

electrodes are present at top (what) and bottom (what)

negative, positive

mass

Polyacrylamide Gel Electrophoresis (PAGE) for Protein Analysis

proteins migrate based on their charge and what

proteins with a ‘native’ what charge will run into the gel

negative

detergent

sodium dodecyl sulfate – SDS is a what

denatures

sodium dodecyl sulfate does what to proteins

one molecule of SDS binds for every how many amino acids

two

no matter what their original charge was, all proteins will now migrate towards the what electrode because of the negative charge of the bound SDS molecule

positive

mass

in an SDS-PAGE gel, proteins migrate based only on their what

Proteins with a what molecular weight migrate faster through the gel

lower

Proteins separated by SDS–PAGE are visualized by staining the gel with a dye such as Coomassie what which binds nonspecifically to all proteins

blue

yield

As purification proceeds, you always lose protein (what goes down) but purification should go up

yield

total amount of activity after purification step/total amount of activity at the start

immune response

coordinated set of interactions among several cell types that occur in response to presence of a foreign substance

antigen

molecule or pathogen capable of eliciting an immune response

epitopes

antigen contains what that immune system ‘sees

antibodies

humoral immune response – B cells make what that recognize the antigen

cellular immune response – which cells engulf and destroy invader

T

Antibodies Have how many Identical Antigen-Binding Sites

2

antibodies

immunoglobulin G (IgG) = major class of what

immunoglobulin G (IgG) = major class of antibodies
– ? polypeptide chains: ? heavy chains and ? light chains

4, 2, 2

disulfide

immunoglobulin G (IgG)
– intra- and intermolecular what bonds

Antigen

Antibodies Bind Tightly and Specifically to what

Generated

Antibodies to Specific Proteins Can Be what

purified

Antibodies to specific proteins can be made by injecting a mammal with a what sample of your protein of interest

produced by injecting a protein into an animal

polyclonal antibodies

made by a population of identical B cells in tissue culture

monoclonal antibodies

contain a mixture of antibodies that recognize different parts of the protein

polyclonal antibodies

made by a population of identical B cells in tissue culture

monoclonal antibodies

antibody

In western blotting or immunoblotting, proteins are separated in an SDS–PAGE gel, transferred to a sheet of polymer, and then detected with a what

Western blot assay

uses antibodies to detect a protein

enzyme

Enzyme-linked immunosorbent assay (ELISA) quantifies the amount of protein present because the antibody is linked to an what whose reaction yields a readily identified colored product

immunoprecipitation

If an antibody exists for a protein of interest, it can be used to quickly purify that protein from a mixture, or tell if it is present in a sample, by what

mass spectrometry

if you have an unknown protein or even a sample of unknown proteins, what can be used to identify the protein(s)

cleaved

the sample is what in some fashion and subjected to mass spectrometry, revealing a series of peptides with known masses

In matrix-assisted laser desorption (MALDI) mass spectrometry, proteins or protein fragments are precipitated onto a matrix and a laser flash releases a pulse of which charged ions from the sample

negatively

time of flight

measures how fast the ions ‘fly’ toward a detector

x-ray crystallography

pattern of diffracted x-rays is collected directly, and an image is reconstructed by mathematical techniques

Electron

X-ray Diffraction Produces what Density Maps from Protein Crystals

Protein Crystallography Steps

1)

X-ray diffraction patterns from protein crystals

Protein Crystallography Steps

2)

three- dimensional electron- density map using a Fourier transform

Protein Crystallography Steps

3)

localized atomic nuclei

Protein Crystallography Steps

4)

completed protein structure

Nuclear Magnetic Resonance

Distances between Protein Atoms Can Be Measured by what

nuclear magnetic resonance

carried out on molecules in solution

molecules

nuclear magnetic resonance captures dynamics of protein structure:
– conformational changes
– interactions with other what

cryo-electron microscopy

sample of the structure of interest is quick-frozen in vitreous (or non-crystalline) ice and kept frozen while being observed in two dimensions with the electron microscope

Cryo-Electron Microscopy

Thousands of Individual Molecules Are Used to Determine Structures by what