Lecture 7

Question 1

HOW ARE PROTEINS ANALYZED?

Answer 1

 Purity – separation from other proteins
 Quantity – how much protein has been purified?
 Activity – has purification/separation maintained protein activity (e.g. enzymes)?

Question 2

What does Electrophoretic analyses

Answer 2

analyze relative amounts of proteins, including protein
of interest
a. Native electrophoresis (agarose or PAGE)
b. Denaturing SDS‐PAGE (enables size determination) & Western blot
c. 2‐D gel electrophoresis

Question 3

What are the Quantitative protein assays and what it used for

Answer 3

measure the total concentration of protein, not just

protein of interest, e.g. Lowry or Bradford protein assays

Question 4

What is activity assays used for

Answer 4

measure the activity of the protein of interest

Question 5

What is Electrophoresis

Answer 5

migration of charged particle in an electric field

Question 6

What is examples of GEL ELECTROPHORESIS

Answer 6

Agarose gel

electrophoresis and Vertical acrylamide gel electrophoresis

Question 7

How does Agarose gel

electrophoresis work

Answer 7

Separation of charged particles:
• Negative molecules towards anode
• Positive molecules towards cathode

Separation of charged particles:
• Negative molecules towards anode
• Positive molecules towards cathode

Separation of native proteins: LDH isoenzymes
Detection enzyme specific reaction:
• Lactate  pyruvate
• Nitroblue tetrazolium  formazan

Question 8

What is Vertical acrylamide gel electrophoresis

Answer 8

Analytical method to separate and visualise
proteins

Can be used to:
• estimate the number of proteins within a mixture
• determine properties such as approximate molecular weight and isoelectric point (2‐D gels – see last
slide)
• determine purity of a protein preparation (i.e. efficacy of purification
process)

• Gel made of polyacrylamide (PAGE)
• Crosslinked polymer (e.g. acrylamide)

Question 9

How does Vertical acrylamide gel electrophoresis operate

Answer 9

• Matrix acts like a molecular sieve (proteins move in proportion to their charge‐to‐mass ratio)
• An electrical field causes the proteins to move down the gel
• Small proteins encounter little resistance as they
move through the gel
• Large proteins encounter greater resistance as
they move through the gel

Question 10

How does protein migrate in the gel

Answer 10

due to size and shape of the molecule

μ = V/E = Z/f

μ = electrophoretic mobility of a molecule
V = velocity of the molecule (affected by charge)
E = electrical potential (force moving the macromolecule)
Z = net charge of the molecule
f = frictional coefficient (in part reflects protein’s shape)

Question 11

How does SDS‐PAGE

(Polyacrylamide gel electrophoresis): Denaturing gel electrophoresis

Answer 11

• SDS contributes large net negative charge  intrinsic protein charge is
negligible
• Proteins are unfolded  all proteins have similar shape (rod liked shape)
Proteins will migrate through polyacrylamide according to mass (size)
Separation according to size depends on the concentration of acrylamide

Question 12

What are the separation range for different Acrylamide concentration (% w/v)

Answer 12

6, 8, 10, 12, 15
Separation range (kDa) 50‒200, 30‒95, 20‒80, 12‒60, 10‒43

Question 13

How do you visualise the protein

Answer 13

by staining

• Coomassie Brilliant blue: dye molecule binds to
protein forming a blue dye‐protein complex
(Detects 50 ng protein in a band)
• Silver stain: proteins bind silver ions, which can then
be reduced under specific conditions to build up a
visible image
(Detects 1 ng protein in a band: VERY SENSITIVE)

Question 14

How can specific protein be identified

Answer 14

Immunoblot (Western blotting)

• Transfer proteins from SDS‐PAGE gel to a nitrocellulose membrane
• Treat the membrane with antibody

Question 15

What are anti-bodies

Answer 15

 Immunoglobulins produced in response to antigens

 Recognise and bind at specific epitopes

Question 16

What is the use of SDS-PAGE

Answer 16

analysis shows only 1 protein present following purification by column chromatography

Question 17

if 1‐D gels have limited ability to resolve proteins in a complex mixture, what other way to resolve proteins

Answer 17

Two‐dimensional gel electrophoresis
can improve resolution
2‐D GE combines SDS‐PAGE with
isoelectric focusing (IEF)
 First dimension: IEF – separation
according to charge
 Second dimension: SDS‐PAGE –
separation according to size

Question 18

What are the advantages of Two‐dimensional gel electrophoresis
can improve resolution

Answer 18

Advantages:
 Sensitive
 Separates proteins with identical Mr but different pI
 Separates proteins with same pI but different Mr

Question 19

How is protein assays done

Answer 19

Using UV Spectrophotometry to determine the amount or concentration of protein in a sample
*Absorbance at 280 nm:
Due to presence of aromatic amino acid residues

Question 20

What is one protein assays

Answer 20

Colorimetric assays
Using dyes and
a standard curve to determine the amount or concentration
of protein in
a sample

Question 21

Two main groups of assays based on chemistry involved for protein assays

Answer 21

 protein‐dye binding chemistry (e.g. Coomassie/Bradford)

 Protein‐copper chelation chemistry (e.g. Lowry)

Question 22

How do you choose which protein assay to use

Answer 22

• Availability of assay

* Compatibility with sample to be analysed

Question 23

What is buiret assay

Answer 23

 Range: 5‒160 mg mL‐1
 Biuret reagent: KOH + CuSO4 plus potassium sodium tartrate (KNaC4H4O6∙4H2O)
 In alkaline conditions protein/polypeptide (two or more peptide bonds) form a complex with copper in the reagent
 Mode of action: Cu2+ forms a coloured coordination complex in an alkaline solution in the presence of
 Proteins: blue to violet
 Short chain polypeptides: blue to pink
 Detection: at 540 nm, absorption of reactive reagent is directly proportional to the concentration of the polypeptide/protein

Question 24

What is Bradford Assay

Answer 24

 Range: 20‒1500 μg mL‐1
 Basis: Spectral shift of Coomassie Brilliant Blue G‐250 dye at low pH (acidic conditions)
 Absorption max of free dye ≈ 465 nm (red form)
 Absorption max when bound to protein ≈ 595 nm
 Absorption of the bound dye is proportional to the amount (concentration) of protein in the sample
(Micro Assay, 1‐10 µg mL‐1)
 Mode of action:
 Red form of dye donates free electrons to ionisable R‐ groups of protein
 Hydrophobic pockets are exposed
 Positive amine groups are positioned in proximity with negative charges on the dye
 Increase in Absorbance at 595 nmis proportional to
bound dye  protein concentration

Question 25

What Lowry assay

Answer 25

 Range: 10‒1000 μg mL‐1
 Detection:
Absorption of reduced Folin reagent (measure at 750 nm)
 Mode of action (not well understood):
 Cu2+ catalyses oxidation of
aromatic amino acids under alkaline conditions
 Phosphomolybdotungstate of the Folin reagent is reduced to heteropolymolybdenum blue
 Resultant blue colour is proportional to tyrosine and tryptophan content of the protein sample
 Is sensitive to pH changes (maintain assay at pH 10‒10.5)
 Use standard curve of known protein amount to compare absorbance of ‘unknown’ protein

Question 26

Best choice of standard protein

Answer 26

pure version of protein to be
analysed or predominant protein in
sample
 Standard curve for Lowry – not linear

Question 27

How do you measure the activity of protein

Answer 27

Measures the activity of
a protein rather than
the actual amount of
a protein.
e.g. Enzyme assay for glucose
6‐phosphate dehydrogenase (G6PDH) – Experiment 6; or enzyme assay for lactate dehydrogenase (LDH):
LDH catalyses the conversion of lactate to pyruvate