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 95C 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 OrganizationPrimary structure = linear chain of amino acids

• Secondary structure = domains of repeating structures, such as β-pleatedsheets 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

-

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

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 Functiontitin 3000 center myosin in sarcomere dystrophin 400 anchoring to plasma membranefilamin 270 cross-link filaments into gel

myosin heavy chain 210 slide filamentsspectrin 265 attach filaments to plasma

membranenebulin 107 regulate actin assembly -actinin 100 bundle filaments gelosin 90 fragment filamentsfimbrin 68 bundle filaments

actin 42 form filaments

tropomyosin 35 strengthen filaments

myosin light chain 27 slide filamentstroponin (T, I, C) 30, 19, 17 mediate regulation of

contractionthymosin 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