Transcript Myosin (light chain)

SEPARATION AND
DETECTION OF PROTEINS
part II
Vlasta Němcová,
Michael Jelínek,
Jan Šrámek
SDS-PAGE
(= sodium dodecylsulphate-polyacrylamide
gel electrophoresis)
- method for separation of proteins according
to their size (molecular weight)
Protocol:
 preparation of polyacrylamide gels
 mixing of tissue lysates with sample buffer
 heating of samples at 95C
 loading of samples onto polyacrylamide gel
 electrophoresis of samples
 staining of the gel with separated proteins in
Coomassie Blue stain
 destaining of the gel
 localization of actin and myosin in the gel,
comparison of their expression in tissues used
 chemiluminiscent detection of actin
Separated proteins in polyacrylamide gel
1
2
3
4
5
6
7
--- Myosin (heavy chain)
--- Actin
--- Tropomyosin
--- Myosin (light chain)
--- Myosin (light chain)
--- Myosin (light chain)
1. Marker
5. Actin
2. Liver
6. Myosin
3. Heart
7. Marker
4. Muscle
What is in sample buffer for SDS-PAGE?
 Tris buffer provides appropriate pH
 SDS (sodium dodecyl sulfate) - detergent that
dissolves proteins and gives them a negative
charge
 glycerol makes samples sink into wells during
loading onto gel
 bromophenol blue is tracking dye moving ahead
of proteins – indicator of separation progress
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
• Quarternaty structure = several polypeptide
chains associated together to form a functional
protein
Preaparation of samples for SDS-PAGE
 before separation proteins have to be denaturated
 denaturation by heating at 95°C in sample buffer
containing SDS
 the proteins no longer have any secondary,
tertiary or quaternary structure
Preaparation of samples for SDS-PAGE
 resultant SDS-coated proteins take on a rod-like
shape and a uniform negative charge-to-mass ratio
proportional to their molecular weights
 speed of protein migration in gel depends ONLY
on their molecular weight
How does an SDS-PAGE
separation work?
• negatively charged
proteins move to positive
electrode
_
+
• smaller proteins move
faster
• proteins are separated by
their size
(molecular weight)
Why use polyakrylamide gels to
separate proteins?
 smaller pore size than agarose
 proteins are much smaller than chromosomes and
routinelly separated fragments of DNA produced in
PCR reaction
 polymerization of acrylamide and N´,N´methylenbisacrylamide is started by reaction
initiator, amonium persulfate (APS), and a catalyst,
tetramethylenediamine (TEMED)
Protein size (molecular weight)

measured in daltons (Da) or kilodaltons (kDa),
1 kDa = 1000 Da

Dalton = atomic mass unit
= 1 Da correspond to mass of hydrogen
molecule (1.66 x 10 -24 g)

average nucleotide pair = 649 Da

average amino acid = 110 Da
200 AA protein – cca 20 kDa
vs.
100 bp fragment DNA – cca 65 kDa
Muscles Contain 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
Separated proteins in polyacrylamide gel
1
2
3
4
5
6
7
--- Myosin (heavy chain)
--- Actin
--- Tropomyosin
--- Myosin (light chain)
--- Myosin (light chain)
--- Myosin (light chain)
1. Marker
5. Actin
2. Liver
6. Myosin
3. Heart
7. Marker
4. Muscle
Western blot analysis
(imunochemical detection of proteins)
 transfer of separated proteins from the gel onto a
membrane
 identification of particular protein by imunodetection
(=binding of primary and secondary antibody)
 visualization by color reaction or chemiluminescence
 the name of the method is a pun of the name SOUTHERN blot, a
technique for DNA detection developed earlier by Edward
Southern
 similarly is named NORTHERN blot, technique for detection of
RNA
heart
heart
equal volume of samples obtained
form separate isolations
heart
liver
muscle
30 ug of protein
heart
actin+myosin
MW
Chemiluminiscent detection of actin
blot of
SDSPAGE gel
Examples of applications of Western blot
in medicine
 detection of antibodies (e.g. for confirmation
of diagnosis)
• boreliosis, EBV, HIV, HSV, Helicobacter pylori
• autoantibodies, antibodies against nuclear antigens
(ANA), antibodies against neural antigens
Principle of tests used in clinical praxis
• Membrane strips containing
electrophoretically separated
antigen extracts are used as solid
phase. The position of the proteins
depends on their respective
molecular masses. .
• If the sample is positive, specific
antibodies in the diluted serum
sample attach to the antigens
coupled to the membrane.
• The attached antibodies react with
AP-labelled anti-human antibodies
(AP = alkaline phosphatase).
Principle of tests used in clinical praxis
• The bound antibodies are stained
with a chromogen/substrate
solution which is capable of
promoting a color reaction. An
intense dark band at the line of
the corresponding antigen
appears if the serum sample
contains specific antibodies.
• Evaluating the band patterns on
the incubated membrane strips
involves differentiating nonspecific from specific antibodies.
The number and intensity of the
specific bands is decisive for the
result "positive/negative".
EUROLINE ANA-Profile 3
Line immunoassay for
confirmation and
discrimination of antibodies
HIV-1 and HIV-2
EUROLINE-WB: detection of
antibodies against Borrelia