Lab Techniques

Wash

-reverse of the extraction process in order to remove unwanted impurities. -Small amount of solute is used to extract and remove impurities

In Vivo and in Vitro

"in vitro" --> outside of normal biological habitat. Think petri dish and test tubes. "in vivo" --> inside of biological habitat. Think clinical trials of drugs.

Isoelectric Focusing

-A gel electrophoresis method that separates proteins on the basis of their relative contents of acidic and basic residues. -The gel has a pH gradient and the proteins will migrate through the gel until they reach the pH that matches their isoelectric point. -At the pI, the protein has a neutral charge, so it will no longer be attracted to the anode and it will stop migrating.

Extraction

-Extraction is a fundamental technique used to isolate one compound from a mixture -done by the transfer of a dissolved compound (desired product) from a starting solvent into a solvent where the product is more soluble -Two solvents must be immiscible - form two layers and do not mix. Nonpolar Layer: Organic layer, dissolves nonpolar compounds. (ether layer) Polar Layer: Aqueous (water) layer. Dissolves compounds with hydrogen bonding or polarity

High Performance Liquid Chromatography (HPLC)

-High-performance liquid chromatography (HPLC), a purification technique ideal for small sample sizes, separates compounds based on polarity. One of two types of columns—normal-phase (NP) or reverse-phase (RP)—may be used. --NP-HPLC consists of a polar stationary phase (typically silica) and a nonpolar mobile phase. --RP-HPLC has a nonpolar stationary phase (typically C18 alkyl hydrocarbon) and a polar mobile phase. -column is usually stationary phase

Chromatography

Separates two or more molecules from a mixture. Stationary Phase: Typically polar. Polar molecules elute slower. Mobile Phase: Typically nonpolar. Nonpolar molecules elute faster.

Vacuum Distillation

Should be used if the boiling points are over 150°C to prevent degradation of the product. The vacuum lowers the air pressure, which decreases BP an ebulliator can be added to introduce small air bubbles into the system. This is the same function provided by a boiling chip at atmospheric pressure. The air bubbles break the surface tension of the liquid being heated and prevent superheating and bumping.

Simple Distillation

Can be used if the boiling points are under 150°C and are at least 25°C apart -Apparatus consists of a: distilling flask (contains the combined liquid), distillation column (thermometer and a condenser), receiving flask (collects distillate).

Reducing SDS-PAGE

Exactly the same as SDS-PAGE, but with the addition of a reducing agent, b-mercaptoethanol, which will reduce the disulfide bridges and result in a completely denatured protein. -lose function

Filtration

-Isolates a solid from a liquid Gravity Filtration: solvents own weight pulls it through the filter and is most commonly used when the filtrate is the desired product. Vacuum filtration: solvent is forced through the filter by a vacuum connected to the flask, used when the solid is the desired product.

Primer

-Must have high GC content and either a G or C at each end. -Example: 5'-GCATAGAAGCATTCCGC-3'

Distillation

-Separates liquids according to differences in their boiling points. -Liquid with the lower boiling point will vaporize first, and the vapors will rise up into the distillation column to condense in a water cooled condenser, then forms a condensate which drips down into the vessel and forms the distillate -The mixture must be heated slowly to ensure that a lower boiling-point compound evaporates before a higher boiling-point compound and that the molecules are separated.

Thin-Layer Chromatography (TLC)

-Sheet coated in polar silica gel. -Molecules are spotted on the bottom of the sheet. -Sheet is placed in a nonpolar liquid. -Mobile phase travels up the plate using capillary action. -Nonpolar molecules have the highest Rf value. (elute faster)

Liquid Chromatography

-Silica is used as the stationary phase while toluene or another nonpolar liquid is used as the mobile phase.

Fractional Distillation

-Used to separate liquids less than 25°C apart to allow more refined separation of liquids by BP -Only the desired product drips down to the receiving flask once it goes through the "rungs" of the column.

Column Chromatography

-Uses an entire column filled with silica or aluminum beads as an adsorbent, which allows for greater separation. -Uses gravity as opposed to capillary action to move the solvent and compounds down the column. Can be used to separate and collect macromolecules such as proteins and nucleic acids (biochemistry)

mass spectrometry

-ionizes molecules in a sample, and the ions can fragment. -ions are accelerated toward a magnet, deflected according to mass, and detected. -A plot of ion mass abundance vs. m/z ratio is generated; -tallest peak = most abundant MASS fragments of the sample can be identified by the m/z difference between two peaks in the mass spectrum.

Sanger DNA Sequencing

-is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. -See more on review sheets

Steps of PCR

1. Denaturation (96°C) 2. Annealing (55 - 65°C) 3. Extension (72°C) -Cycle is repeated until you have enough DNA

Polymerase Chain Reaction

A method of producing thousands of copies of DNA segment using the enzyme DNA polymerase -The key ingredients of PCR are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). -The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.

SDS-PAGE

A polyacrylamide gel electrophoresis method for proteins using DENATURING conditions. -Sodium Dodecyl Sulfate denatures the proteins and gives the proteins a uniform charge. -This allows them to be separated solely on mass, thus, you can estimate the protein's molecular mass. -lose function -most mobility = smallest weight

Native-PAGE

A polyacrylamide gel electrophoresis method for proteins using NON-DENATURING conditions. -Proteins keep their native charge and structure so they are separated based on charge and size.

Gel-Filtration Chromatography (Size-exclusion)

Separates molecules by size rather than polarity. Smaller molecules enter the porous gel beads allowing them to elute later. Larger molecules will elute faster because they do not fit in the pores and will not be slowed down.

Loading Control

Proteins that are ubiquitously expressed (ie, they have consistent concentration levels across all cell/tissue types) are used as loading controls

Affinity Chromatography

Separates proteins based on their affinity for a specific ligand. (key word TAG) -Beads are bound to a specific ligand and proteins with a high affinity for that ligand will bind to the beads. -Proteins with a low affinity for the ligand will elute first.

Ion Exchange Chromatography

Separates proteins by their net charge. -The column is filled with charged beads, either POS or NEG. Cation Exchange: NEG beads used, NEG proteins elute 1st. (POS elute farthest) Anion Exchange: POS beads used, POS proteins elute 1st. (NEG elute farthest) (anionic peptides bind to anion-exchange columns

Taq Polymerase

The DNA polymerase typically used in PCR. Named after the heat-tolerant bacterium from which it is isolated (Thermos aquaticus). -Very heat-stable and most active around 70°C

Gel Electrophoresis

The separation of nucleic acids or proteins, on the basis of their size and electrical charge, by measuring their rate of movement through an electrical field in a gel. -For proteins and small molecules the gel is polyacrylamide. -For larger molecules (>500 bp), the gel is agarose. -Negatively charged molecules travel toward the anode at the bottom. -Large molecules will move SLOWER.

Recrystallization

Used to further purify crystals in solution Product is dissolved in a minimum amount of hot solvent and is cooled down to let it recrystallize. Solvent should be chosen so that the solute (product) is only soluble at high temperatures

Resolving Agent

a chiral compound used for separating enantiomers.

Gas (Liquid) Chromatography

separates compounds in a mixture based on boiling point. -mobile phase is an inert gas -stationary phase is a liquid that coats the column, which is in a heated oven. -molecule with the lowest boiling point (lowest molecular weight) will reach the detector first