Tools of Protein Biochemistry

Question 1

Electrophoresis

Answer 1

Native-no denature-charge and size

SDS-linearlize and coat in (-) charge-just size

MOVE FROM (-) to (+)

Question 2

Western

Answer 2

Gel, paper (ntirocell is (+)) with probe

Inject protein of interest into org to get antibody

1st degree protein of interest, to 2nd degree antibody to 1st degree protein, with indicating enzyme attached (third degree)

Get color change where protein is

Question 3

Epitope

Answer 3

Portion of molecule which antibody binds to
-sugar, lipids, aa’s

tags-added to molecule to visualize

Question 4

Indicator

Answer 4

Antibody with bound enzyme that catalyzes conversion of colorless molecule to colored
ex. horseradish/alkaline phosphatase

Question 5

HRP parthway

Answer 5

horseradish phophatase

HRP on antibody, H2O2 binds to HRP, DAB added, when in contact-H2O2 falls off and makes color

use nickel sulfite to make darker

Question 6

ELISA

Answer 6

Detects antibodies/antigens-measures concentration of them (can be used to measure concentration proteins)

direct=1st degree labaled, indirect=second degree labeled

1. antibody to antigen is applied to sample
2. binding detected by direct/indirect
3. Add substrate to get color

Small amount of sample, high sensitivity (false + rate high)

in wells can put antigen or antibody only (from what i saw so far)

Question 7

HIV TESTING

Answer 7

ELISA with western blot confirmation
-window period where virus is not being able to be seen 6 weeks

see p31 or p24 or p160 or p120-postive

then do western/wait and do western

only tests for antibody for HIV (antigens on plate)

Question 8

Acute MI

Answer 8

thrombus causes ischemia (no blood supply)

Troponin-contractile regulatory protein os straited muscle
-isoforms Ta din increase after MI
ELISA-use immobile antibody for cTroponin (cardiac troponin)

Question 9

CoIP/Immunochrom

Answer 9

Discover macromolecular interactions between proteins

want to isolate proteins together

FIX THIS CARD

Question 10

Immunoaffinity chrom steps and why

Answer 10

Loading on column

Want to isolate interacting proteins

Get 1 protein

Put antibody to protein on column (get from injected org)

ellute, harsh wash

see what got

if not too harsh-can find enzymatic activities

Question 11

Immunohistochem

Answer 11

Put antibody on antigen you want to study

Have another antibody to that antibody with indicator enzyme on it (like HRP)

Shows color-tissue which antigen is located

Question 12

Mdm2 and P53

Answer 12

Mdm2 is activated under cellular stress and ubi p53

-found with co-ip

Question 13

Her-2

Answer 13

protmotes proliferation of breast cells

-excess Her-2=high risk for breast cancer

Question 14

Epitope tagging

Answer 14

add an AA tag

Have antibody to tag

If see antibody know we have whatever was tagged

visualize what tissue protein is in and interactions