Lecture 14: Techniques and Technology

What are David’s 3 levels of science?

-What we know (memorizing facts)

-How we know what we know

-Asking new questions, seeking answers, making new hypotheses, and testing them

How do biochemists identify and study proteins?

purifies the proteins involved and studies their behavior in vitro; recapitulates the process in a test tube

How do geneticists identify and study proteins?

isolate a mutant that is defective in a particular process

How do cell biologists identify and study proteins?

watches the process in a microscope, follows the dynamics of particular proteins in a living cell, inhibits the function of a specific protein in a living cell, and blocks the process their interested in

How are proteins purified?

cell extraction, centrifugation, and fractionation

How are cells extracted?

tissues or cells are placed in buffer to break cells open

What does centrifugation do?

separates soluble from insoluble materials (separates supernatant from the precipitate)

What is fractionation?

proteins are separated based on their physical and chemical properties

What is column chromatography?

the column is filled with beads that will separate proteins in the cell extract based on certain properties

What are the different types of column chromatography?

ion-exchange and gel-filtration chromatography

What are some characteristics of ion-exchange chromatography?

Beads have chemical groups attached to them

Proteins with the opposite charge of the beads will attach to the column

In column chromatography, how are proteins eluted from the column?

the concentration of the salt in the buffer is increased, and the salt will compete with the protein for bead binding

What are some characteristics of gel-filtration chromatography?

Separates proteins based on size

Beads have different size tunnels

Large proteins will go around the beads and will elute quickly from the column

Medium-sized proteins will fit in some tunnels but not all and will elute off the column more slowly

Smaller proteins will take the longest to elute from the column

How do researchers identify the fractions that contain the protein of interest?

an in-vitro assay is performed

What is an assay?

a biochemical test/reaction

True or False: Different proteins need different assays.

true

What is involved in an in-vitro assay?

DNA (plasmid), protein fraction, and radioactive nucleotides

True or False: In vitro assay, fractions containing RNAP make RNA polymers, and fractions lacking RNAP cannot make RNA polymers.

true

True or False: Radioactivity in the RNA polymer precipitates in the presence of TCA.

true

True or False: Repeated rounds of purification in combination with an assay allow purification of specific proteins of interest.

true

What do fractions refer to?

partially-purified proteins

True or False: Knowing a protein’s structure often provides insight into its mechanism of action.

true

What is chaperonin?

a protein folding device; helps other proteins fold properly

What is important to know about chaperonins?

Made up of hydrophobic groups that stick out in the inner chamber

Provides a unique and new chemical environment compared to the cytoplasm for a protein to fold properly

What does DNA helicase do?

separates dsDNA into sdDNA

What are some characteristics of DNA helicase?

Is a hexamer with a hole in middle to allow for ssDNA to pass through but not dsDNA

Uses ATP hydrolysis

How is a protein’s structure determined?

x-ray crystallography

What is important to know about X-ray crystallography?

Crystals are difficult to form

Electrons in the protein will diffract x-ray beams

Calculations need to be made to reveal the structure

Reveals an electron density map

True or False: The structure of a protein cannot be determined without knowing it’s amino acid sequence. With the electron density map, the known size and shape of the amino acids can be optimally to the electron density map to reveal the structure of the protein of interest.

true

What is immunofluorescence used for?

to visualize the localization or dynamic properties of a particular protein within a cell

What is the basic process of immunofluorescence?

A reagent is added that binds only to the protein of interest (an antibody)

The reagent will contain a fluorescent chemical group that will allow us to light up the protein of interest

What is an advantage of immunofluorescence?

only the protein of interest is seen (everything else is black)

How are antibodies “made”?

antibodies are purified from the blood

Each B cell makes how many antibodies?

only 1 type of antibody

True or False: Antibodies purified from live animals contain a mixture of antibodies, and each of these antibodies recognize a different epitope.

true

What is an epitope?

the location where antibodies recognize and bind to antigens

What are polyclonals?

a mixture of the different antibodies that are purified from the blood

What is a limitation of polyclonal antibodies?

the limited supply of them

What is an advantage of polyclonals?

they can recognize multiple epitopes; at least 1 antibody will bind to the target protein

In monoclonal antibodies, what happens to the B cells?

individual B cells are isolated

What are 2 limitations of monoclonal antibodies?

Researchers cannot grow B cells in the lab

Antibody may not bind under all conditions

How do researchers produce monoclonal antibodies?

immortal cancer B cells are fused with B cells

What is the structure of an antibody?

2 heavy chains and 2 light chains

Variable regions (differs from antibody to antibody)

Constant region

What region of an antibody binds to the epitope?

the variable region

How are antibodies labeled?

radioactivity, a fluorescent tag, and chemical reactions (enzymes are heated)

What is most common way to label antibodies?

through fluorescent tagging

What are the two types of antibodies?

primary and secondary antibodies

What is the primary antibody?

the antibody directed against the protein of interest

What is the secondary antibody?

marker-coupled antibodies directed against the primary antibody

True or False: Secondary antibodies come from a different organism than the primary antibody.

true

Where do secondary antibodies bind?

to the constant region of the primary antibody

Is the primary or secondary antibody fluorescently tagged?

the secondary antibody is tagged

How is the fluorscent protein visualized?

through fluorescence microscopy

What are the 3 barriers of fluorescence microscopy?

First barrier filter: lets through only blue light with a wavelength between 450 and 490 nm

Beam-splitting mirror: reflects light below 510 nm, but transmits light above 510 nm

Second barrier filter: cuts out unwanted fluorescent signals and passes specific green fluorescein emission between 520 and 560 nm

What light hits the anibody and emits a green light?

blue light

In fluorescence microscopy, what color(s) is seen?

green

What dye is used to visualize microtubules?

DAPI

True or False: In pictures with 2 fluorescent colors, the pictures must be superimposed on top of each other.

true

True or False: Some antibodies bind proteins in such a way that they do not inhibit the function of that protein, others bind in such a way that they do inhibit the function of the protein.

true

What are “blocking” antibodies?

antibodies that inhibit the function of a protein

What are 3 different techniques for inserting DNA into cells?

transfection, transformation, and transegenics

What are some characteristics of transfection?

Transient expression

Transfect cultured cells by lipid treatment or electroporation

Protein is expressed from cDNA in plasmid DNA

DNA does not last very long (typically 2-3 days)

Typically done in tissue cultural

True or False: It’s difficult to make plasmids in human cells; the DNA will be broken down.

true

What are some characteristics of transformation?

Used for stable, long-term expression

Transfect cultured cells by lipid treatment or electroporation

Select for a particular antibody resistance

Protein is expressed from cDNA integrated into the host chromosome

More difficult to perform this experiment than a transfection

Typically done in tissue culture

What are some characteristics of transfenics?

Integrates a gene into the genome in the gametes of an organism

All progeny in subsequent generations will contain that gene in all cells

True or False: Transformation, transfection, and transgenics differ in the fate of the DNA that is inserted into the cells/organism.

true

What are some uses of transformation, transfection, and transgenics?

Puts mutant forms of genes into cells

Can visualize cells and proteins via GFP labeling and fluorescence microscopy

Where is GFP found?

in jellyfish

True or False: Tissue specific promoters that drive GFP transcription allow specific cell types to be imaged in transgenic organisms.

true

What do GFP fusion proteins allow for?

subcellular localaization of a protein of interest

GFP is attached to which end of the protein of interest?

the CTD end

What is DAPI?

a dye that fluoresces blue when bound to DNA but not when unbound

What does GFP allow for?

the imaging of live cells

What does antibody staining typically involve?

fixed (deadly) cells

What is the goal of cryoelectron microscopy?

to determine the structure of proteins, especially large protein complexes

Where is classic electron microscopy performed?

in a vacuum without any water

In the absence of water, what can protein do?

adopt altered conformations (non-proper conformations)

What does cryoelectron microscopy allow for?

high resolution electron microscopy in an aqueous environment

What happens to proteins in cryoelectron microscopy?

samples are rapidly frozen and viewed at very low temperatures to preserve native protein conformations; 100,000’s of molecules in various orientations are sorted

What type of proteins do cyroelectron microscopy work well for?

large molecular complexes such as ribosomes