Transcript Prezentace aplikace PowerPoint

5. SEPARATION
AND DETECTION OF
PROTEINS II
SDS-PAGE
Jana Vobořilová,
Anna Kotrbová-Kozak,
Vlasta Fürstová,
Tereza Kopská
SDS-PAGE
(= sodium dodecylsulphate-polyacrylamide
gel electrophoresis)
-method for separation of proteins according
to their molecular weight
Outline of second part of the
experiment
*Prepare polyacrylamide gels
*Add diluted samples to the sample buffer
*Heat to 95C for 4 minutes
*Load the samples onto polyacrylamide gel
*Run 200 volts for 30-40 minutes
*Stain in Coomassie Blue stain
*Destain
*Identify molecular markers, actin and
myosin in the separated proteins
Levels of Protein Organization
Primary structure = linear chain of
amino acids
• Secondary structure = domains of
repeating structures, such as β-pleated
sheets and α-helices
• Tertiary structure = 3-dimensional
shape of a folded polypeptide,
maintained by disulfide bonds,
electrostatic interactions, hydrophobic
effects
• Quaternary structure = several
polypeptide chains associated together
to form a functional protein
-proteins denatured by heating
them in a sample buffer containing
sodium dodecyl sulphate (SDS)
-the proteins no longer have any
secondary, tertiary or quaternary
structure
-resultant proteins take on a rod-like
shape and a uniform negative charge-tomass ratio proportional to their molecular
weights
Migration of such proteins in
electric field:
-negatively charged proteins move
towards the positive pole
-migration of proteins:
*directly proportional to the overall
charge of proteins
*inversely proportional to protein
size (molecular weight)
How does an SDS-PAGE gel
work?
•Negatively charged
proteins move to
positive electrode
s-s
SDS, heat
•Smaller proteins
move faster
• Proteins separate
by size
proteins with
SDS
-
+
What is in the Sample Buffer?
*Tris buffer to provide appropriate pH
*SDS (sodium dodecyl sulphate) detergent to
dissolve proteins and give them a negative
charge
*Glycerol to make samples sink into wells
*Bromophenol Blue dye to visualize samples
SDS-Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
CH3
CH2
•SDS (Sodium Dodecyl
Sulfate) detergent
CH2
CH2
CH2
CH2
–solubilizes and
denatures proteins
CH2
CH2
CH2
–negative charge to
proteins
CH2
CH2
CH2
O
O
O
-
O
•Heat denatures proteins
S
SDS
Why Use Acrylamide Gels to
Separate Proteins?

Acrylamide gel: tight matrix

Ideal for protein separation

Smaller pore size than agarose

Proteins much smaller than intact
chromosonal DNA
– average amino acid = 110 Da
Protein Size

Size measured in daltons (Da) or kilodaltons (kDa)

Dalton
= atomic mass unit
= corresponds to mass of hydrogen
molecule (1.66 x 10 -24 gram)
= defined also as 1/16 of the mass of an
atom of oxygen

Average amino acid = 110 Da
Average nucleotide pair = 649 Da
Gel Analysis
Lane
1. Kaleidoscope Markers
2. Shark
3. Salmon
4. Trout
5. Catfish
6. Sturgeon
7. Actin and Myosin Standard
Muscle Contains Proteins of
Many Sizes
Protein
kDa
Function
titin
dystrophin
filamin
3000
400
270
center myosin in sarcomere
anchoring to plasma membrane
cross-link filaments into gel
myosin heavy chain
210
slide filaments
spectrin
membrane
nebulin
a-actinin
gelosin
fimbrin
265
attach filaments to plasma
107
100
90
68
regulate actin assembly
bundle filaments
fragment filaments
bundle filaments
actin
42
form filaments
tropomyosin
35
strengthen filaments
myosin light chain
27
slide filaments
troponin (T, I, C)
contraction
thymosin
30, 19, 17
mediate regulation of
5
sequester actin monomers
Extension of the study
WESTERN Blot Analysis
*transfer of separated proteins from the gel
onto a membrane
*identification of a protein by a complex of
primary and secondary antibodies
*visualization by color reaction or by
chemiluminiscence
WESTERN blot (method for
detection of protein):
-its name is a pun off the name Southern
blot, a technique for DNA detection
developed earlier by Edward Southern
-similarly is named Northern blot,
method for detection of RNA