gel electrophoresis

Question 1

the purpose of using electrophoresis is to?

Answer 1

separate proteins, DNA, or RNA based on size and/or charge

Question 2

the gel types of are made out of either?

Answer 2

acrylamide or agarose

Question 3

acrylamide or agarose are used because they form?

Answer 3

nets as it solidifies

Question 4

how are the pores (nets) effected when more acrylamide or agarose is used in the initial solution?

Answer 4

the more acrylamide or agarose, the smaller the pores in the nets

Question 5

the bottom of the plate mold is what charge?

Answer 5

the positively charged anode

Question 6

the top of the plate mold is what charge?

Answer 6

the negatively charged cathode

Question 7

since voltage/ current is being applied the gel, it acts as a?

Answer 7

electrolytic cell and has the same principles

Question 8

what size molecules have a harder time moving towards the positive anode?

Answer 8

larger ones because of the gel resistance and pore size

Question 9

what size molecules migrate the fastest?

Answer 9

smaller ones because they fit easily through the pores

Question 10

the ladder is a lane of a collection of?

Answer 10

molecules where we know the size to be compared to the unknown samples

Question 11

in step 1: the sample DNA (or RNA or protein) is?

Answer 11

isolated from cells

Question 12

in step 2: restriction endonucleases enzyme is sometimes used in order to?

Answer 12

cleave the strands of DNA into smaller fragments of varying size

Question 13

in step 3: a loading dye is added to the DNA sample in order to?

Answer 13

make the loaded sample more visible and inhibit DNA degradation

Question 14

glycerol in the loading dye makes the sample more?

Answer 14

dense than the surrounding buffer thus means the DNA will sink to the bottom of the gel wells

Question 15

in step 4: the mixture of fragments are loaded into cell wells and the electrical current is ran in order to?

Answer 15

migrate the samples to the positive end

Question 16

in step 5: the bands are DNA are visualized by?

Answer 16

adding a dye that binds to nucleic acids and fluoresces them when exposed to UV light

Question 17

electrophoresed gel can be transferred into more solid and stable membrane, which is a process called?

Answer 17

blotting

Question 18

native-PAGE is a method that uses polyacrylamide gel for proteins and occurs under what conditions?

Answer 18

under non-denaturing conditions so that it will separate proteins by size while remaining their structure

Question 19

SDS-PAGE is a method that uses polyacrylamide gel for proteins under what conditions?

Answer 19

under denaturing conditions in order to separate proteins by mass

Question 20

SDS-PAGE works by adding negatively charged sodium dodecyl sulfate (SDS) to solution of proteins which does what?

Answer 20

denatures them so that they bind to one SDS for every two amino acids, giving the same charge-to-mass ratio for all thus they can be separated purely on mass

Question 21

in SDS-page, the smallest proteins are found?

Answer 21

towards the bottom

Question 22

SDS-PAGE cannot interrupt with what types of bonds?

Answer 22

covalent thus if disulfide bonds are present they will not be broken

Question 23

reducing SDS-PAGE is a method the exact same as SDS-PAGE but adds what to do what?

Answer 23

adds a reducing agent (ex: beta-mercaptoethanol) to reduce disulfide bridges so the protein will be completely denatured

Question 24

isoelectric focusing is a method that separates proteins based on?

Answer 24

their acidic and basic residues

Question 25

in isoelectric focusing, a polyacrylamide gel is made with pH gradient so that the protein will migrate to the anode in order to determine?

Answer 25

the protein’s isoelectric point - when the protein is at it’s pI, the net charge is zero thus the protein will not be attracted to the positively charged anode and will not move