BIOL 1406 Lab Exam 2

Enzyme

an organic molecule (usually a protein) that speeds up a chemical reaction

Catalyst

a molecule that speeds up a chemical reaction but is not consumed or permanently altered by the reaction

factor

Any _______that affects the interaction between an enzyme and its substrate(s) will affect enzyme activity.

Factors affecting enzyme activity

• Temperature

• pH

• Substrate concentration

Chromatography

a technique used to separate molecules that are in a mixture

means “color writing” and was originally used to separate pigments from plant extracts

Stationary phase

the coating of silica on a TLC plate, which is a rectangular strip of plastic

Mobile phase

a mixture of two nonpolar organic solvents: petroleum ether and acetone

charged

In thin layer chromatography, ______ pigments will appear as slowly migrating horizontal bands

less charged/uncharged

In thin layer chromatography, ______ pigments will appear as faster migrating horizontal bands

solvent front

leading edge of the solvent on TLC plate that indicates how far the solvent has traveled during chromatography

not enough spinach extract applied to plate

The finished TLC plate has pigment bands that appear very faint. What might have caused this result?

the solvent level was too high or origin was too low

There are no pigment bands on the finished TLC plate. However, the solvent at the bottom of the developing chamber is very brightly colored. What might have caused this result?

Rf

the migration of a pigment band along the TLC plate relative to the migration of the mobile phase (organic solvent mixture)

unitless and range from 0 to 1

Rf equation

Distance from origin to pigment band

Distance from origin to solvent front

smaller Rf

Pigments that are more attracted to the stationary phase will travel more slowly away from the origin. Thus these pigment bands will have a ________.

larger Rf

Pigments that are more attracted to the mobile phase will travel farther from the origin. Thus these pigment bands will have a _________.

Polar compounds

make strong interactions with the silica gel and therefore move at a slower rate

Nonpolar compounds

move farther with the solvent because they are weakly absorbed by the stationary phase

Absorption spectrum

measures the relative amount of light a molecule absorbs within a range of light wavelengths

Absorption maximum

Each major peak in the absorption spectrum

Size exclusion chromatography

a technique often used to isolate specific molecules from a molecule mixture

also called column chromatography

Column matrix

in SEC, a plastic tube filled with the column material (ex: sephadex beads)

Larger proteins

_________ will elute from the column faster than smaller ones in SEC.

Parallel dilution formula

C₁V₁ = C₂V₂

Electrophoresis

the process of placing molecules in a porous matrix (gel) and applying an electrical current to move them through the matrix

used to separate molecules according to size, shape, and charge

Polyacrylamide gels

frequently used for electrophoresis because of their ability to separate molecules like proteins or DNA

made from cross-linked acrylamide molecules

Native protein

a protein that retains its natural shape

Protein denaturing solution

dismantles protein shapes and ensures that all proteins have a net negative charge

Beta-mercaptoethanol

a denaturing agent that breaks disulfide bonds found in protein tertiary structures

Sodium dodecyl sulfate

this a detergent that denatures proteins, giving them the same negative charge so that the only variable affecting them during electrophoresis is their size

Coomassie blue

a stain used after electrophoresis to completely visualize the originally colorless protein bands on polyacrylamide gel

Protein marker

a mixture of proteins with known molecular weights to assist in the calculation of the molecular weights of other proteins

Cathode end

the well end of the gel

negatively charged

Anode end

the bottom end of the gel

positively charged

Denature the protein and eliminate disulfide bonds so that migration is only affected by molecular weight.

What is the purpose of boiling your protein samples before loading them into the polyacrylamide gel?

Yes because same molecular weight

Would you expect GFP and BFP to migrate the same distance during electrophoresis? Why?

The denatured protein is no longer a dimer and has a smaller size than native GFP, which is still an intact dimer and larger in size.

Explain why the molecular weights of native and denatured GFP are different.

Genetically modified organism

an organism with DNA that has been deliberately altered to accelerate its growth or improve its pest and disease resistance

Promoter

DNA sequence that ensures transcription will be initiated properly by RNA polymerase

Terminator

DNA sequence that acts as a stop signal for RNA polymerase

Polymerase chain reaction

molecular biology technique used to clone or make identical copies of a specific DNA sequence

PCR distinct features

• occurs in a tube instead of a living cell

• copies a specific target DNA sequence instead of all of the DNA in a cell

• uses different temps instead of different proteins for most of DNA replication

• primers are single-stranded DNA not RNA

Template DNA

in PCR, the source of the target sequence

Primers

short sequences of single-stranded DNA that are designed to flank the target sequence that is to be copied

DNA polymerase

will bind to the primers and extend them to copy the target DNA sequence

DNA nucleotides

mixture of individual nucleotides that will eventually be linked together by DNA polymerase to make the copies of the target sequence

Buffer

provides the ideal pH and salt concentration for PCR

Denaturing step

the reaction is heated to a high temperature to break the covalent bonds holding the base pairs of the template DNA strands together

occurs at 94 C

Annealing step

Single stranded DNA primers base pair with template DNA

occurs at 59 C

Extension step

Replication by DNA polymerase of the template DNA to make a complementary strand by extending the primer

occurs at 72 C

Plant PCR

This reaction contained primers designed to target a DNA sequence that should be found in all plant DNA

GMO PCR

This reaction contained primers designed to target two possible DNA sequences only present if the food contained DNA from genetically modified plants

Nucleic acids

_________ have a net negative charge, so electrical current will cause the DNA in a sample to migrate out of the well toward the anode (+) end of the gel.