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 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-to- mass 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 Smaller proteins move faster Proteins separate by size - + s-s SDS, heat 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) SDS (Sodium Dodecyl Sulfate) detergent –solubilizes and denatures proteins –negative charge to proteins Heat denatures proteins O S O O O - CH 2 CH 3 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 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 ProteinkDaFunction titin3000center myosin in sarcomere dystrophin400anchoring to plasma membrane filamin270cross-link filaments into gel myosin heavy chain 210slide filaments spectrin 265attach filaments to plasma membrane nebulin107regulate actin assembly  -actinin100bundle filaments gelosin90fragment filaments fimbrin68bundle filaments actin42form filaments tropomyosin35strengthen filaments myosin light chain 27slide filaments troponin (T, I, C)30, 19, 17 mediate regulation of contraction thymosin5sequester 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