L7 Protein Purification

Question 1

What does 6x His tag allow

Answer 1

purification of the GFP protein from other bacterial proteins

Question 2

Histidine residues bind to _ ions including nickel, cobalt, and copper under specific buffer cond

Answer 2

metal

Question 3

Affinity purification

Answer 3

Beaded agarose or magnetic particles with chelating groups can immobilize metal ions, which functions as ligands for binding and purification of 6xHis-tagged proteins

Question 4

What buffer conditions are best for the binding of Ni-agarose to 6xHis tag?

Answer 4

near-neutral 7.2

Question 5

Low concentration of imidazole helps to prevent

Answer 5

nonspecific binding of endogenous proteins that have histidine clusters

Question 6

Imidazole is an ____ in the binding buffer

Answer 6

advantage

Question 7

Why is it good to have imidazole in the binding buffer?

Answer 7

To remove contaminants while allowing His-tag protein binding

Question 8

In affinity protein purification of His-tagged protein, why would you elute with imidazole?

Answer 8

To compete with the His-tagged protein for binding to the Ni-agarose beads

Question 9

What is an advantage of size and shape gel filtration?

Answer 9

conditions can be varied to suit the sample or requirements for further processing
Good for molecules who may be sensitive to pH, ions, or harsh environmental conditions

Question 10

Ion exchange chromatography IEX

Answer 10

Reversible interactions b/w charged molecules and oppositely charged IEX media are controlled in order to favor binding or elution of specific molecules and achieve separation

Question 11

Due to Tyr and Trp residues, proteins have a characteristic absorption at

Answer 11

275-280nm

Question 12

What impact could DNA have on the measurement of protein

Answer 12

We could overestimate [DNA] b/c DNA also contains aromatic rings

Question 13

Bradford assay involves binding of ___ to ___

Answer 13

Coomasie blue to proteins

Question 14

Under acidic cond the dye is__

Answer 14

red

Question 15

When the dye binds the protein it is converted to a ___ form

Answer 15

blue

Question 16

The blue protein-dye is detected at ___nm in the assay using a ____

Answer 16

595nm, spectrophotometer

Question 17

What is the ideal way to select a protein standard?

Answer 17

A purified prep of the protein you are assaying - absolute reference std

Question 18

If you cannot use a purified prep you can

Answer 18

use a relative standard which would need to be experimentally determined

Question 19

What are the 4 protein assays

Answer 19

Enzyme activity
Binding assay
Immunological
Bioassay

Question 20

Enzyme activity

Answer 20

measuring how much of a protein is required to digest a standard amount of substrate

Question 21

Binding assay

Answer 21

Receptors might be assayed by its ability to bind ligands, which could be labeled by radioactivity or fluorescence

Question 22

Immunological technique

Answer 22

Antibodies with highly specific affinity to certain proteins can be used in techniques to detect immobilized portiens

Question 23

Bioassays

Answer 23

To detect specific biological acitivty

Question 24

If acrylamide is less concentrated, there is ___ cross-linking and the more ___ a given protein migrates

Answer 24

less, rapidly