1. SEPARATION AND DETECTION OF PROTEINS part II Vlasta Němcová, Michael Jelínek, Jan Šrámek

2. SDS-PAGE (= sodium dodecylsulphate-polyacrylamide gel electrophoresis) - method for separation of proteins according to their size (molecular weight)

3. 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

4. Separated proteins in polyacrylamide gel
--- Myosin (heavy chain)
--- Actin
--- Tropomyosin
--- Myosin (light chain)
--- Myosin (light chain)
--- Myosin (light chain)
1. Marker Actin
2. Liver Myosin
3. Heart Marker
4. Muscle

5. 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

6. 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

7. 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

8. 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

9. _ + 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) _ +

10. 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)

11. 100 bp fragment DNA – cca 65 kDa
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 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

12. Muscles Contain Proteins of Many Sizes
Protein kDa Function
titin center myosin in sarcomere
dystrophin anchoring to plasma membrane
filamin cross-link filaments into gel
myosin heavy chain 210 slide filaments
spectrin attach filaments to plasma membrane
nebulin regulate actin assembly
a-actinin bundle filaments
gelosin fragment filaments
fimbrin bundle filaments
actin form filaments
tropomyosin 35 strengthen filaments
myosin light chain 27 slide filaments
troponin (T, I, C) 30, 19, 17 mediate regulation of contraction
thymosin 5 sequester actin monomers

13. Separated proteins in polyacrylamide gel
--- Myosin (heavy chain)
--- Actin
--- Tropomyosin
--- Myosin (light chain)
--- Myosin (light chain)
--- Myosin (light chain)
1. Marker Actin
2. Liver Myosin
3. Heart Marker
4. Muscle

14. 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

15. Chemiluminiscent detection of actin
equal volume of samples obtained form separate isolations MW
actin+myosin
heart
muscle
liver
heart
heart
heart
30 ug of protein
blot of SDS-PAGE gel

16. 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

17. 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).

18. 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 non-specific from specific antibodies. The number and intensity of the specific bands is decisive for the result "positive/negative".

19. EUROLINE ANA-Profile 3
EUROLINE-WB: detection of antibodies against Borrelia
Line immunoassay for confirmation and discrimination of antibodies HIV-1 and HIV-2