7.protein purification

Question 1

what is the most common salt used for the fractionation of proteins by salting out?

Answer 1

ammonium sulphate (NH4)2SO4

Question 2

which method is used for protein denaturation?

Answer 2

salting out

Question 3

the principle of dialysis is based on

Answer 3

separation of proteins by their size

Question 4

the dialysis tube is made up of

Answer 4

cellophane

Question 5

in dialysis, The size of the particle being removed depends on the..

Answer 5

quality of dialysis membrane (pore size), and the quality of both the particle and the solvent.

Question 6

If the blood plasma is dialysed against such an isotonic solution, which contains no Na+ ions…

Answer 6

only the Na+ ions will pass through the membrane into the external fluid.

Question 7

if we fil NaPr solution in a dyalisis tube and dialyse it against NaCl solution, what will happen?

Answer 7

NaCl will pass through the membrane in the direction of NaPr solution, because from the aspect of Cl– concentration this latter solution is less concentrated.

Question 8

in which method do we use Nessler’s reagent?

Answer 8

dialysis

Question 9

what is the Nessler’s reagent?

Answer 9

alkaline solution of potassium-tetraiodomercurate(II)

Question 10

what will be the color of the dialysis product? why?

Answer 10

yellow-brown

in alkaline milieu, NH3 is released, which forms yellow brown coloured mercury(II)-oxide-amido-iodide; HgO.Hg(NH

Question 11

the method of gel filtration is based on

Answer 11

separation based on size (molecular mass)

Question 12

The most frequently used commercially available materials for gel filtration are the

Answer 12

Sephadex gels.

Question 13

what is the elution volume in dialysis?

Answer 13

the volume of eluent required to cause elution

Question 14

which has more eluted volume? (small proteins, large, negative, positive)?

Answer 14

small proteins

Question 15

in which method do we use Dextran-blue solution?

Answer 15

in gel filtration

Question 16

in which method do we use Bromophenol red?

Answer 16

in gel filtration

Question 17

the method of electrophoresis is based on

Answer 17

separation of proteins based by their charge

Question 18

which elution silution do we use in gel filtration?

Answer 18

NaCl solution, flow rate: 2ml/minute

Question 19

in the electrophoresis, which proteins have side groups and easily can bind a proton?

Answer 19

lysine and arginine

Question 20

in the electrophoresis, which proteins can easily lose a proton?

Answer 20

aspartic acid and glutamic acid

Question 21

what is the isoelectric point (electrophoresis)?

Answer 21

That pH value, at which (due to the emergence of zwitterionic structure) the number of positive and negative charges is equal (the molecule is neutral to the outer milieu, the netto electric charge is zero)

Question 22

what determine the isoelectric point?

Answer 22

the side chain of an amino acid

Question 23

At the pH below the isoelectric point the protein has a … charge, while above it a … charge.

Answer 23

positive

negative

Question 24

Most of the globular proteins have an isoelectric point between

Answer 24

pH 4.5-6.5

Question 25

which medium do we use nowadays in electrophoresis?

Answer 25

primarily agarose-, polyacrylamide- or starch gel

Question 26

The most current electrophoresis procedure is the so called

Answer 26

SDS-PAGE (SDS = Sodium dodecylsulphate, PAGE = polyacrylamide gelelectrophoresis).

Question 27

what is the role of mercaptoethanol?

Answer 27

cleaves the disulfide bridges which stabilizing the native structure of the proteins

Question 28

during electrophoresis, SDS - anionic detergent (negative) migrate towards the

Answer 28

positive anode

Question 29

after electrophoresis, the separated proteins can be made visible by staining the gel with

Answer 29

coomassie brilliant blue

Question 30

in which method is it possible to separate the isoenzymes from different tissues, for example in the case of lactate dehydrogenase (LDH)?

Answer 30

staining the gel after electrophoresis (with coomassie brilliant blue)

Question 31

when staining the gel after the electrophoresis, the separated proteins can be transported to

Answer 31

a nitrocellulose membrane (elecrtic separation. This process is called blotting and the whole method is called Western blot.)

Question 32

The advantage of blotting is that the ….

Answer 32

membrane (in contrast of the gel) can be incubated with primary antibodies, specific for the examined protein. The binding of these primary antibodies can be made detectable by labelled secondary antibodies.

Question 33

The secondary antibodies that used to incubate the primary antibodies in the membrane of the blotting method are usually conjugated with the so-called…..

Answer 33

horseradish peroxide enzyme which forms (after adding the proper substrate and hydrogen peroxide) a product, detectable in UV-light. So the produced bands specifically indicate the given protein.