Lab Quiz 2

Question 1

limit for passaging

Answer 1

3 months

Question 2

freezing down tissue cells

Answer 2

500 microL MDCK cells in media and 500 microL DMSO

Question 3

polyclonal antibodies

Answer 3

can bind to multiple sites

Question 4

monoclonal antibodies

Answer 4

only one site gets bound

Question 5

polyclonal adjuvant

Answer 5

allows for the slow release of antigen from injection site

Question 6

polyclonal antibody titer

Answer 6

antibody concentration (want high)

Question 7

polyclonal agglutination experiment

Answer 7

“clumping” looks at titer

Question 8

polyclonal affinity purification

Answer 8

use Protein A or G

Question 9

Monoclonal: Step One

Answer 9

spleen extracted, red blood cells lysted, B-cells separated using density gradient centrifugation

Question 10

Monoclonal: Step Two

Answer 10

B-cells then fused to myeloma cells

Question 11

Monoclonal: Step Three

Answer 11

get rid of myeloma + myeloma cells by using denovo or salvage pathways

Question 12

What is used to fuse B-cells to myeloma cells

Answer 12

polyethylene glycol

Question 13

B-cell + B-cell

Answer 13

will die out

Question 14

B-cell + myeloma

Answer 14

want

Question 15

myeloma + myeloma

Answer 15

don’t want

Question 16

nucleotide synthesis

Answer 16

denovo or salvage pathways

Question 17

Salvage Pathway

Answer 17

knock our HGPRT enzyme in myeloma cells

Question 18

Denovo Pathway

Answer 18

knock out by placing HAT media-Aminopterin

Question 19

Aminopterin

Answer 19

inhibits denovo pathway

Question 20

Monoclonal: Step Four

Answer 20

sample dilluted; cloning cylinders placed over and one cell added to new plate

Question 21

Immunoprecipitation

Answer 21

small scale affinity purification of an antigen using a specific antibody

Question 22

Monoclonal vs. Polyclonal

Answer 22

monoclonal: expensive longer; polyclonal: cheaper, shorter, multiple epitopes to amplify signal, flexibility

Question 23

Downside of Polyclonal

Answer 23

each batch is different and could cross react

Question 24

Protein of Interest

Answer 24

Zo-1 (found in tight junction)

Question 25

Lysis (detergent) solution

Answer 25

0.5M EDTA at pH 8, 1.0M Tris at pH 8, Deoxycholate, NP-40

Question 26

EDTA

Answer 26

chelates Ca and Mg

Question 27

Tris

Answer 27

buffer to keep from denaturing proteins

Question 28

deoxycholate

Answer 28

ionic (harsh)

Question 29

NP-40

Answer 29

nonionic (non-harsh

Question 30

lysis buffer requirements

Answer 30

stabilize native protein conformation; inhibit enzymati activity by pH

Question 31

Protein A or G or combo

Answer 31

aid to actual precipitation; on sepharose beads

Question 32

sepharose beads

Answer 32

cyanogen bromide to covalently link to beads

Question 33

Protein A

Answer 33

S. aureus (4 binding sites)

Question 34

Protein G

Answer 34

Streptococci (2 binding sites and more broad); cell wall proteins, but binds to heavy chain of antibody via Fc region

Question 35

Western Blot

Answer 35

acrylamide with bisacrylamide to form a crosslinked polymer network; ammonium persulfate added; TEMED (tetramethylenediamine) for a catalyst

Question 36

Running Buffer

Answer 36

tris, Glycine, SDS

Question 37

tris and Glycine

Answer 37

keeps everything moving uniformly

Question 38

SDS

Answer 38

keeps everything linearlized

Question 39

Stacking Gel

Answer 39

on top; lower concenration of acrylamide; pH 6.8

Question 40

Resolving Gel

Answer 40

bottom; higher concentration of acrylamide; pH 8.8