Protein purification and analysis

Question 1

How to create a mixture for protein purification ?

Answer 1

Must homogenise to break the cell open

Question 2

How to create a homogenate ?

Answer 2

1. sonification
2. french press
3. douncer

Question 3

1st step to creatng homogenate ?

Answer 3

Centrifugation from slowest speed to fastest speed heaviest pellet will form
first
applying force that induces seperation based on specific mass or density
1. nuclei
2. mitochondria
3. microsomal

Question 4

How to simplfy the mixture ?

Answer 4

Salting out as many proteins are less soluble when the solution has a higher salt concentration
by adding ammonium sulphate
as proteins have different conc at which they precipitate from solution
with crude mixture-used to reduce complexity of solution

Question 5

What is a seperation method ?

Answer 5

Chromotography used to seperate molecules based on their chemical and physical characteristics

Question 6

Define statiionary phase ?

Answer 6

solid,tend to remain certain molecules

Question 7

Define mobile phase ?

Answer 7

solvent tends to elute certain others

Question 8

What are factors affecting the binding ?

Answer 8

ph
ionic bonding
temp
solvents

Question 9

What are 2 diff types of chromotography ?

Answer 9

Thin layer-cellulose,silica
Column -sepharose

Question 10

How does chromatogrophy work?

Answer 10

tubes filled with beads of approprate material -usually inert
chemically derivatised to alter properties
can use UV to detect protein exiting the column and automated collectors to move the tes tube

Question 11

What is spectrophotmetry ?

Answer 11

Method to measure the concentration of a compound
Uses as a parameter the absorbance value of a standard solution at a given wavelength
The level of absorbance is proportional to the concentration of that compound in the solution

Question 12

What are chemical charectertics of protein ?

Answer 12

7 side chains can ionise
terminal amino and carboxyl groups canalso ionise
some side chains-very hydrophobic
so proteins can have different pl(pH which no net surface charge )

Question 13

What are the types of column chroatography ?

Answer 13

1. Size exclusion
2. ion exchange
3. affinity

Question 14

How does size exclusion work ?

Answer 14

Small molecules enter the bead in space between the polymer chains
large molecules bypass the beads and come out first
can also use analytically -run set proteins of known size through the column and compare the elution time to sample

Question 15

How do the samller molecuels move down slower ?

Answer 15

beads are porous
percolate down the mixture
small molecules enters beads slower
large molecules flow through faster
UV spectrophotometry to detect the protein exiting the column

Question 16

How does ion exchange work ?

Answer 16

chargwed side chains are on the surface of the soluble proteins
if proteins has a net charge this can be used to column material
elute the protein by adding a lot of salt -the ions outcompete the binding of proteins to gel
cn have +ve or-ve colums
can also elute pH charge

Question 17

How does affinity chromatography work ?

Answer 17

Some proteins bind small molecules that can be linked to the beads
Can be used for antibody purification - use the antigen
Elute by disrupting binding
add excess ligand
change pH
Often, microbes are genetically engineered to produce a protein fused to such a ‘tag’.
e.g. maltose binding protein

Question 18

What is SDS page ?

Answer 18

sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
The proteins become denatured (unstructured) and covered with SDS
Electrophoresis used to force the proteins to migrate through a gel
This gel is made of polyacrylamide.
Visualize the proteins - use a blue dye called Coomassie

Question 19

How does gel electrophoresis work?

Answer 19

We can use gels and electric charge again to separate molecules
A charged molecule will move in an electric field - electrophoresis
A gel offers resistance to movement
How fast a molecule moves is proportional to net charge and inversely proportional to mass
If we make the proteins have a uniform charge per mass, then we can separate on size only due to resistance from the gel
small ones will migrate faster than larger ones

Question 20

How does the quantificaion of proteins work ?

Answer 20

Bradford assay
A280 measurements