MCBL/ Protien Isolation

what are proteins?

large complex molceules in our body that are reuireed for structure, function & regulation of body tissue

What is protein isolation ?

involves seperating protein molecules from a complex structure using purifcation technqiues

proteins are seperated by

1)salt precipation (solubillity)

2) Ion exchange (charge)

3) SDS (size)

4) western blot analysis

What is the enzyme we isolated?

rubisco

what is rubsico?

plant enzyme responsible for oxygen and carbon

rubsico is made of 2 polypeptide subunits, what are they?

8 large subunits 55kda (coded in chlorplast), 8 small subunits 14kda(coded in nucleus)

what is the preciapte in this experiment?

ammonium sulfate- and it recovers molecules from mixtures

Salting in

low concentration of salt increase proteins solubillity

Salting out

high concentration of salt decrease leading to precipation

whats the point of dialsysis?

removes unwanted molecules like salt from solution

what % is salting in?

37% saturation/ *210g/L

what % is salting out?

50% // * 85g/L

steps in ammonium sulfate precipation?

salt in and salt out

buffer 1

used for homegenizing spinach

what are the 3 componets of rubisco ?

1)high solubillty 2) high negative charge 3)2 polypeptides 55-14

buffer A

A salt gradient is used to elute separated proteins? t/f

trueee

Ion exchnage chromtopgrpaghy?

Separation technique that purifies molecules based on their charge or ionic
interactions

neg charge - cation or anion?

cation

pos charge - cation or anion?

anions

are the beads pos or neg?

pos

What are the steps in ion exhcange?

1)equailibration 2)sample amplifciation 3)elution 4)regeneration

what is equailibration?

stablizes the psotive charges on resin

whats sample amplifcation?

Binding of proteins to the resin.

what is elution?

Removal of bound protein by applying buffers with
varying salt concentrations

what is regeneration?

removes molecules still bounds, and regenerates the colum for the next run.

what does the sandwhich for protein trasnfer consist of?

Casset, sponge, filter paper, membrane, gel, filter paper, sponge

sds seperates based on ?

size

what does sds stand for?

Sodium Dodecyl Sulphate
Polyacrylamide Gel
Electrophoresis

what type of gel is used?

Acrylamide and Bis-acrylamide

sds has Pre-cast gel (vertical set-up) t/f?

trueee

what reduces disulfide bridges?

β-mercaptoethanol or dithiothreitol–

what effects the sample buffer for color and density ?

Bromophenol blue and Glycerol

whats western blot?

Used for the identification of proteins
within a cell or tissue lysate

whats the princiaple of western blot?

Based on the binding of
antibodies on specific epitopes on the
protein (s) of interest to identify the
target protein within the lysate.

what are anitbodies?

proteins that bind to antigens or other proteins

which types of antibodies for western blot ?

Primary and secondary antibodies

primary antibodies?

binds to the anitgen of protein interest

secondary anitbodies ?

binds to primary

what is the reported enzyme that produces color or light,
which allows it to be easily
detected and imaged

Alkaline phosphatase

Ion exchange why??

we used ion charge to target rubisco because its negative charge

Why do we heat sample

to disrupt tertiary structures of proteins