BMB 251H Exam 4

Centrifugation

Separating cellular components from each other based on size (density)

Centrifuging smaller particles

Needs a faster spin

Lysed cell homogenate

What goes into centrifugation

Components of a centrifuge

Armored chamber, rotor, refrigeration, motor, vacuum, sedimenting material

Force on contents of centrifuge tube

Varies with rotation speed

Can be separated by centrifugation

Organelles and macromolecules

Types of centrifuge

Fixed angle and swinging bucket

S in 30S, 70S

Sedimentation

Function of gradients in a centrifuge

Small and large biomolecules can be separated based on their mass and shape

Sucrose gradient

Allows for differentiation based on speed of sedimenting

Percoll gradient

Used in immunoassays

Composition of amino acids

Affects the ability of proteins to stick to different surfaces (resins), can be exploited to separate proteins from each other

Other properties of proteins

Can be used to separate proteins

Protein shapes

Globular, filament

Protein sizes

Large, small

Protein accessible charges

No charge, positive, negative

Protein ligands

Binds a specific molecule

Column chromatography

Used to separate proteins

How column chromatography works

Solid matrix in a tube with a porous plug at the bottom, sample is applied to the top, solvent continuously applied to the top forces the sample through and fractionates it, each type of fractionated molecule can be collected from the bottom one by one

Ion Exchange (IEX) Chromatography

Another way to separate proteins based on their charge

During IEX chromatography

Proteins bind with beads that have the opposite charge of their exposed amino acids, allowing proteins with the same charge to flow through

pH change

More damaging way to remove bound proteins in IEX chromatography

Using salt gradient

Gentler way to remove bound proteins in IEX chromatography

Gel filtration/size exclusion chromatography

Separates proteins based on size

How gel filtration works

Small molecules get stuck in the pores of porous beads, and flow through slower than larger molecules that don’t get stuck

Affinity chromatography

Separates proteins based on their affinity for a ligand

How affinity chromatography works

Ligand is attached to beads, protein that binds to the ligand is captured and the rest is washed away, elute off the bound protein by breaking the interaction between it and its ligand

DNA affinity chromatography

If using DNA of many different sequences, will result in many different DNA-binding proteins being captured. If using specific DNA sequence, will only capture rare protein that specifically recognizes the sequence.

Protein purification

Successively passing samples containing the protein activity over different columns in sequence

Purifying a sample

Removes contaminating proteins but loses material at every step

Protein activity

Defined by the scientist, can be ability to bind a ligand or ability to catalyze a reaction

Standardized purification

Molecular biologists use epitope tags to accomplish this

Epitope tags

Tag is inserted into the DNA that encodes a protein, and is then used to quickly elute out that protein and any associated ones

Types of epitope tags

6xHis (Nickel-NTA-Agarose), Glutathione-S-Transferase (Glutathione-Agarose), Maltose Binding Protein (Amylose-Agarose), Protein A (IgG Antibody-Agarose), Calmodulin Binding Peptide (Ca++/Calmodulin-Agarose)

Tandem Affinity Purification Tag

Can use more than one epitope tag on a protein, each tag is for a different affinity column

Gel Electrophoresis

Used to characterize preparations of biomolecules

How gel electrophoresis works

Negatively charged molecules move from top to bottom (negative to positive), and their motility will be affected by their size and shape

Polyacrylamide Gels

A mixture of macromolecules travels through a porous gel at different speeds

SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE)

A method to characterize the composition of samples that separates molecules based upon their mass, not their shape or charge, larger proteins remain near the top

Sodium Dodecyl Sulfate (SDS)

One of two key reagents for SDS-PAGE, denatures proteins and coats the polypeptide chain, giving it a negative charge

Beta-Mercaptoethanol (2-ME)

One of two key reagents for SDS-PAGE, breaks disulfide bonds

Dyes

Such as Coomassie Blue or Silver Stain, allow us to see proteins on a gel

Mass spectrometry

Used to determine what protein is what in a complex mixture

Another way to identify proteins in a mixture

Cut proteins up using a site-specific protein, separate peptides by column chromatography, fragment the peptides and use to identify order of amino acids and post-translational modifications

Trypsin

Site-specific protease that cuts before R or K

Antibodies

Used in many assays for proteins because antibodies can be selected that bind specific proteins

Antigen

The molecule used in the immunization (e.g. protein, nucleic acid, small molecules)

Epitope

The part of the antigen that is recognized by an antibody

Antibodies in nature

Proteins produced be a vertebrate immune system that recognize foreign molecules which have entered the body

Monoclonal antibody

An isolated antibody that binds a single epitope on one antigen

Polyclonal antibodies

A mixture of antibodies that together bind many epitopes on one antigen

Raising specific antibodies to a protein-of-interest

Immunize an animal, antibodies are produced in the animal, antibodies are secreted into blood, blood is collected from the animal, blood is centrifuged to separate plasma from cells, column chromatography is used to purify antibodies

Primary vs secondary antibody

Primary antibody binds to the antigen, multiple secondary antibodies can bind to the primary antibody, allowing for multiple reporters to bind to the secondary antibodies

Western blotting

Proteins are transferred from gel to membrane through electroelution, membrane is covered with a blocking agent that prevents non-specific binding, membrane is incubated in solution that has primary antibody, membrane is washed to remove non-specific binding, membrane is incubated in solution that has secondary antibody

Molecular Cloning (Recombinant DNA Technology, Genetic Engineering, DNA Cloning)

Cut-and-paste with DNA, DNA molecules can be cut into fragments with Restriction Endonucleases, then pasted together using a DNA ligase to seal the backbones

Restriction Endonucleases (Restriction Enzymes)

Bacteria produce these to protect themselves against invasive DNA, they bind a specific DNA sequence, cut both strands, and leave blunt ends or ends with an overhang, hundreds are available

Shorter recognition sequences for REs

Occur by chance more frequently than longer sequences

Recombinant DNA Molecules

Product of ligating together DNA fragments with compatible ends

Generating a recombinant DNA molecule

A plasmid is cleaved with a RE, the DNA fragment to be cloned is ligated in, the plasmid is introduced into a bacterial cell, the cell is replicated, copies of the plasmid can be isolated from lysed bacteria

Plasmid

Circular DNA, contains an origin of replication and a gene that acts as a selection marker (often antibiotic resistance)

Selection marker

Helps make sure that the plasmid being replicated is the correct one, ex. plasmid has antibiotic resistance, cell is cultured in antibiotic to force retention of the plasmid

Libraries

A collection of all nucleic acid sequences in a genome

Genomic DNA (gDNA) libraries

Contains all DNA in genome, including regulatory regions, introns, nontranscribed DNA, etc.

Complementary DNA (cDNA) libraries

Only expressed sequences in the genome, first goes through transcription, then RNA splicing to result in mRNA, then produces cDNA copies of the mRNA

Gel electrophoresis and DNA

Can be used to characterize DNA, large DNA migrates more slowly, used to isolate specific DNA fragments and to characterize a population of DNA molecules produced in an experiment

Agarose gel

Polymer of carbohydrate, DNA fragments are often separated on it

Northern Blotting

Detects RNA

Southern Blotting

Detects DNA

Northern and Southern blotting

Nucleic acids separated by gel electrophoresis, blotted onto paper, fragments are hybridized to radiolabeled probe, visualized by autoradiography

Nucleic acid hybridization

Highly specific binding between two strands with sequence homology, how nucleic acids are detected in complex mixtures: using a labeled probe, can visualize the location and amount of DNA on a blot or in a cell by detecting the probe

DNA synthesis reactions

Can create a probe used in hybridization experiments, takes a purified DNA restriction fragment, denatures and anneals it with a mixture of hexanucleotides, adds DNA polymerase and labeled nucleotides, the polymerase incorporates the labeled nucleotides

Polymerase Chain Reaction (PCR)

A common way to isolate a rare specific DNA fragment from a mixture or amplify sequences of a gene

How PCR works

Heat is used to separate strands, they’re cooled to anneal primers, materials are added and DNA synthesis occurs (scientist selects primers so some sequence info is needed, but this is readily available for organisms with sequenced genomes)

Problem with PCR

High temperatures denatured the DNA polymerase, requiring fresh enzyme to be put in after every cylce

Thermus aquaticus

Has a heat-stable DNA polymerase that is now used in PCR

Templates for PCR

DNA: genomic clone, RNA: complementary DNA (cDNA) clone

Expression systems

Can be used to express a specific protein at a high level in organisms, protein-coding DNA sequence is inserted into an expression vector which is then introduced into cells, then the sequence results in an overexpression of mRNA and proteins

Advantages of expression systems

Easier to grow bacteria/yeast than specific cells-of-interest, can overexpress protein-of-interest to make a lot of it then can be used for medical, industrial, research purposes

Most often used for overexpression

E. coli, though yeast, insect cells, mammalian cells, and wheat germ extracts can be used too (some eukaryotic proteins don’t fold properly in bacteria or they need to be modified by eukaryotic properties)

Dideoxy (Sanger) sequencing

A form of DNA sequencing that uses chain-terminating nucleotides called dideoxyribonucleoside triphosphates to make partial copies of the DNA fragment to be sequenced

Dideoxyribonucleoside triphosphate (ddNTP)

Lacks the 3’ hydroxyl (OH) group, blocks further elongation of a DNA strand when incorporated into that strand

Automated Dideoxy Sequencing

Uses an excess of normal dNTPs plus a mixture of four different ddNTPs, each labeled with a different color fluorescent tag, results in DNA products that are separated by electrophoresis and identified by the color of the ddNTP they contain

Shotgun sequencing

Used to sequence whole genomes that are small and lack repetitive DNA, the genomic DNA is fragmented and a genomic library is constructed, many clones are sequenced and then the full genome is reconstructed by stitching together the genomic sequence of each clone, using overlaps between clones as a guide

2nd Generation Sequencing: Illumina Platform

Fluorescent marker incorporated into DNA, a photo is taken, the fluorescent marker is removed, next nucleotide is added, repeat. To use with RNA, must first isolate mRNA and convert into cDNA

3rd Generation Sequencing: Nanopore Sequencing

Uses the disruption in an electrical current as a nucleotide goes through a nanopore to identify the nucleotide, can be used with RNA

The Promise of the $1000 Genome

Much easier to have your entire DNA blueprint because it’s much cheaper and there are better methodologies, may be able to have specific drugs/therapies made for you

Limitations of having your entire genomic sequence

Needs time, we don’t know what much of the genome does

Benefits of having your entire genomic sequence

Know your genetic risks

Risks of having your entire genomic sequence

Privacy (insurance companies, etc.), faulty interpretations

Forward genomic screen

Broadly change the genes and select the phenotype you want to study

Methods for forward genetic screening

Chemical mutagen (hit the genome with a DNA-damaging agent), transposable elements (pieces of DNA that can insert semi-randomly with the help of proteins encoded by it), natural sequence variations (sequence many members of a population and correlate with disease traits)

Reverse genetic screen

Select the gene you want to modify and broadly observe for phenotypes that may result

Methods for reverse genetic screening

Introduce a mutation (integrate a different copy of the gene in the normal genomic locus), delete/knock out the gene (integrate a piece of DNA lacking the gene into the normal genomic locus), add another copy of the gene (integrate the correct or variant gene into a different genomic locus), knock down the mRNA or protein (use methods to cause specific mRNA or protein degradation)

Genomic locus

The site of the gene in the genome

Forward Genetic Screen: Temperature Sensitive Proteins in Yeast

Temperature sensitive mutants allow for the identification of essential genes

Reverse Genetic Screen: Amino Acid Substitutions in Recombinant Proteins

Change the sequence of DNA in the test tube and insert the altered DNA (now coding for the desired amino acid) back into the cell

Cells in culture

Populations of cells can be grown in culture dishes, can treat cultured cells in ways that are not possible with whole animals

Primary cells (cell strains)

Vertebrate cells isolated from tissues that have limited proliferative potential

Cell lines

Derived from cancer cells, they proliferate indefinitely, results from genetic changes

Diameter of a typical animal cell

10-20 microns