Lab Techniques

gel electrophoresis

• separation of proteins, DNA or RNA based on size and/or charge

• bottom is the positively charged anode; top is the negatively charged cathode

• negatively charged particles will travel toward the anode

native-page

• nonreducing conditions

• separate proteins while retaining structure

sds-page

• denaturing conditions; SDS denatures the proteins and binds every two amino acids, giving all proteins the same charge:mass ratio

• separate proteins by mass

• only interrupts covalent bonds, so disulfide bonds will not be broken

reducing sds-page

• similar to sds-page

• addition of a reducing agent will reduce disulfide bridges, resulting in a completely denatured protein

isoelectric focusing

• gel electrophoresis method

• separates proteins based on relative contents of acidic and basic residues via a pH gradient

• proteins will migrate through the gel until the pH of the gel matches their isolectric point

western blotting

• (protein)

• proteins are separated based on size via an sds-page gel

• proteins from the gel are transferred to a polymer sheet and exposed to a radiolabeled antibody specific to protein of interest

• polymer sheet is visualized using autoradiography, making the protein of interest visisble

southern and northern blotting

• (dna) and (rna)

• exposed to restriction enzymes to cut DNA into smaller fragments

• fragments are denatured to create single-stranded DNA

• undergo gel electrophoresis

• DNA is transferred to a nitrocellulose paper and exposed to a radiolabeled 32-phosphate DNA probe that is complementary to DNA of interest

• audiography is used to identify the strand of interest

dna sequencing

• determine the sequence of nucleotides in a DNA strand

• DNA strand of interest is denatured to create single-stranded DNA

• ssDNA strand of interest is added to a solution containing: radiolabeled DNA primer complementary to gene of interest, DNA polymerase, all four dNTPs, and small quantity of ddNTP

• gel is transferred to a polymer sheet, and audiography is used to identify the strands in the gel

chromatography (general)

• typically polar stationary phase and non-polar mobile phase

• polar molecules are separated by staying with the stationary phase, while non-polar molecules stay with the mobile phase

liquid chromatography

• silica = stationary phase

• tolulene, or other non-polar liquids = mobile phase

high-performance liquid chromatography (hplc)

utilizes high pressures to pass the solvent phase through a finely-ground stationary phase, giving hplc higher resolving power

gas chromatography

• separate and analyze molecules that can be vaporized

• mobile phase = inert, unreactive gas

• allows polar molecules to elute slower (higher retention time)

size exclusion chromatography

• separate molecules by size

• smaller molecules can enter the porous gel beads, allowing them to elute later

• stationary phase = gel beads

ion-exchange chromatography

• separate proteins by net charge

• cation exchange: negatively-charged proteins will elute first

• anion exchange: positively-charged proteins will elute first

affinity chromatography

• separate proteins based on affinity for a specific ligand

• proteins will low affinity will elute first

thin-layer chromatography

• using capillary action, allows molecule to move with the mobile phase if they are non-polar

• visualized using UV light

• Rf = distance traveld by molecule/distance traveled by solvent front

simple distillation

separate molecules when their boiling points differ by 25 degrees C or greater

fractional distillation

separate molecules when their boiling points differ by less than 25 degrees C

polymerase chain reaction

• amplify a small quantity of DNA by several orders of magnitude

• initially heated to 95C to separate the strands in the presence of complementary DNA primers

• abruptly cooled to 54C to allow primers to anneal to each ssDNA

• new complementary strands are synthesized using Taq DNA polymerase at 72C

• cycle is repeated until desired amount of DNA is synthesized

1H NMR: 0-5 ppm

alkane

1H NMR: 3-5 ppm

alkane with a heteroatom

1H NMR: 5-7 ppm

alkene

1H NMR: 6-8 ppm

aromatic

1H NMR: 9-10 ppm

aldehyde

1H NMR: 10-13 ppm

carboxylic acid

13C NMR: 0-70 ppm

alkane

13C NMR: 90-120 ppm

alkene

13C NMR: 110-160 ppm

aromatic

13C NMR: 160-200 ppm

carbonyl

IR: 1700-1750

• carbonyls

• 1720-1740: aldehydes

• 1700-1725: ketones

• 1735-1750: esters

• 1700-1725: carboxylic acids

IR: 3200-3600

OH groups

IR: 3300-3400

• amines

• number of peaks relative to the number of hydrogens on the amine

immunoprecipitation

• protein of interest is precipitated by adding a bead-conjugated antibody that is specific to the protein of interest

radioimmunoassay

• used to determine the concentration of a protein of interest in a given sample

enzyme-linked immunoabsorbent assay (elisa)

• used to identify the concentration of a molecule of interest in a given sample

• uses primary antibodies specific to the molecule of interest, secondary antibiotics specific to the primary antibiotics and conjugated with a fluorophore (presence measured via spectrophotometry)

edman degradation

• sequence amino acid residues in a protein

• PTH is added to the N-terminal of a polypeptide, leaving an intact polypeptide shortened by one residue

• chromatographic techniques can be used to identify the amino acid

• limitation: can only identify polypeptides less than 50 residues

gram staining

gram positive: appears purple; thick peptidoglycan layer

gram negative: appears pink; think peptidoglycan later sandwiched between two lipid bilayers

tollen’s test

• tests for the presence of an aldehyde (or alpha-hydroxy-ketones)

• can distinguish between aldoses and ketoses

• positive test is characterized by the precipitation of elemental silver

benedict’s test

tests for the presence of an aldehyde (or alpha-hydroxy-ketones)

positive test is characterized by a change in color from clear blue to brick red with the formation of precipitate

fehling’s test

tests for the presence of an aldehyde (or alpha-hydroxy-ketones)

positive test is characterized by a change in color from clear blue to brick red with the formation of precipitate