Lecture 7 Flashcards
HOW ARE PROTEINS ANALYZED?
Purity – separation from other proteins
Quantity – how much protein has been purified?
Activity – has purification/separation maintained protein activity (e.g. enzymes)?
What does Electrophoretic analyses
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
What are the Quantitative protein assays and what it used for
measure the total concentration of protein, not just
protein of interest, e.g. Lowry or Bradford protein assays
What is activity assays used for
measure the activity of the protein of interest
What is Electrophoresis
migration of charged particle in an electric field
What is examples of GEL ELECTROPHORESIS
Agarose gel
electrophoresis and Vertical acrylamide gel electrophoresis
How does Agarose gel
electrophoresis work
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
What is Vertical acrylamide gel electrophoresis
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)
How does Vertical acrylamide gel electrophoresis operate
• 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
How does protein migrate in the gel
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)
How does SDS‐PAGE
(Polyacrylamide gel electrophoresis): Denaturing gel electrophoresis
• 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
What are the separation range for different Acrylamide concentration (% w/v)
6, 8, 10, 12, 15 Separation range (kDa) 50‒200, 30‒95, 20‒80, 12‒60, 10‒43
How do you visualise the protein
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)
How can specific protein be identified
Immunoblot (Western blotting)
- Transfer proteins from SDS‐PAGE gel to a nitrocellulose membrane
- Treat the membrane with antibody
What are anti-bodies
Immunoglobulins produced in response to antigens
Recognise and bind at specific epitopes
What is the use of SDS-PAGE
analysis shows only 1 protein present following purification by column chromatography
if 1‐D gels have limited ability to resolve proteins in a complex mixture, what other way to resolve proteins
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
What are the advantages of Two‐dimensional gel electrophoresis
can improve resolution
Advantages:
Sensitive
Separates proteins with identical Mr but different pI
Separates proteins with same pI but different Mr
How is protein assays done
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
What is one protein assays
Colorimetric assays Using dyes and a standard curve to determine the amount or concentration of protein in a sample
Two main groups of assays based on chemistry involved for protein assays
protein‐dye binding chemistry (e.g. Coomassie/Bradford)
Protein‐copper chelation chemistry (e.g. Lowry)
How do you choose which protein assay to use
- Availability of assay
* Compatibility with sample to be analysed
What is buiret assay
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
What is Bradford Assay
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
What Lowry assay
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
Best choice of standard protein
pure version of protein to be
analysed or predominant protein in
sample
Standard curve for Lowry – not linear
How do you measure the activity of protein
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
