Separation of proteins using SDS page

Question 1

what does SDS page stand for

Answer 1

Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis

Question 2

what is electrophoresis

Answer 2

is the process of moving
charged molecules in solution by applying an
electrical field.

Question 3

the extent a molecule is moved in the electrophoresis depends on? x3

Answer 3

size, shape and charge

Question 4

the polyacrylamide acts as a ?

Answer 4

a molecular sieve and
add a component of size selectivity to the
electrophoresis

Question 5

PAGE - GEL ELECTROPHORESIS- stands for? what kind of gels are used and how are protein bands separated [comment on the matrix it has] and visualised

Answer 5

polyacrylamide gel electrop.
vertical gels are used
separated based on the basis of relative molecular weight- porous matrix acts as molecular sieve
visualised using stains

Question 6

what does SDS do to the protein

Answer 6

its a denaturant and it causes the protein to unfold [linearized] as it disrupts the H bonds and the hydrophobic interactions

Question 7

SDS is a x and has a y charge

Answer 7

detergent and has a negative charge

Question 8

SDS binds -> the

Answer 8

unfolded proteins and gives them overall neg charge as it mask the proteins own charge

Question 9

what is used [finally] to break disulphide linkages in the loading dye

Answer 9

a reducing agent i.e. Beta-mercaptoethanol

Question 10

smaller proteins are on - concentration gels whilst larger proteins = run on - con. gels
range of concentrations

Answer 10

higher
lower
4->18%

Question 11

what are the 5 steps in electrophoresis procedure

Answer 11

1. loading
2. running the gel e.g 85V
3. opening the cast
4. washing/staining/washing
5. visualizing the gel

Question 12

initially, the mobility of the protein is in the middle between

Answer 12

leading buffer ion in the stacking gel [chlorine ion from buffer] and the trailing buffer [glycine ion ] from the upper gel tank

Question 13

proteins enter the stacking gel between these two ion fronts this does what to the protein

Answer 13

Concentrate the protein into a tight stack

Question 14

when the ionic front reaches the separating gel the pH changes in the gel from x -> y

Answer 14

6.8-> 8.8

Question 15

the glycine becomes? charge and the proteins become the x ions and they do ?

Answer 15

negatively charged
trailing ions
unstack themselves according -> their molecular weight as they resolve in the separating gel

Question 16

after the electrophoresis the proteins have resolved into x band and can be visualised using a x stain
what stain binds to protein
this staining method is sensitive to x microgram quantities of proteins
greater sensitivity can be achieved using?
the approx. molecular weight can be calculated by comparing ?

Answer 16

discreet band
Coomassie stain
Coomassie Brilliant Blue R250 binds -> protein
0.1micrograms
silver-staining- 1nanograms
compare the distance travelled by the protein of interest -> set of standard proteins

Question 17

western blotting - proteins separated can be transferred -> a x i.e
the bands can now be analysed by ?

Answer 17

membrane i.e. nitrocellulose
immunophenotyping blotting
- allows selective detection of a protein by a specific antibody and is frequently used in research