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5. SEPARATION AND DETECTION OF PROTEINS II SDS-PAGE Jana Vobořilová, Anna Kotrbová-Kozak, Vlasta Fürstová, Tereza PowerPoint Presentation
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5. SEPARATION AND DETECTION OF PROTEINS II SDS-PAGE Jana Vobořilová, Anna Kotrbová-Kozak, Vlasta Fürstová, Tereza - PowerPoint PPT Presentation


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5. SEPARATION AND DETECTION OF PROTEINS II SDS-PAGE Jana Vobořilová, Anna Kotrbová-Kozak, Vlasta Fürstová, Tereza Kopská. SDS-PAGE ( = sodium dodecylsu lph ate-polyacrylamide gel electrophoresis) -met hod for separation of protein s according to their molecular weight.

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5. SEPARATION AND DETECTION OF PROTEINS IISDS-PAGEJana Vobořilová,Anna Kotrbová-Kozak,Vlasta Fürstová,Tereza Kopská
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SDS-PAGE(= sodium dodecylsulphate-polyacrylamide gel electrophoresis)-methodfor separation of proteinsaccording to their molecular weight

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Outline of second part of the experiment*Prepare polyacrylamide gels*Add diluted samples to the samplebuffer*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
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

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-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

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-resultant proteins take on a rod-like shape and a uniform negative charge-to-mass ratio proportional to their molecular weights
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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 (molecularweight)

how does an sds page gel work

s-s

SDS, heat

-

+

proteins with

SDS

How does an SDS-PAGE gel work?
  • Negatively charged proteins move to positive electrode
  • Smaller proteins move faster
  • Proteins separate by size
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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

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

O

O

O

S

-

O

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
  • SDS (Sodium Dodecyl Sulfate) detergent
    • solubilizes and denatures proteins
    • negative charge to proteins
  • Heat denatures proteins

SDS

why use acrylamide gels to separate proteins
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
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
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
Muscle Contains Proteins of Many Sizes

ProteinkDaFunction

titin 3000 center myosin in sarcomere

dystrophin 400 anchoring to plasma membrane

filamin 270 cross-link filaments into gel

myosin heavy chain 210slide filaments

spectrin 265 attach filaments to plasma membrane

nebulin 107 regulate actin assembly

a-actinin 100 bundle filaments

gelosin 90 fragment filaments

fimbrin 68 bundle filaments

actin42form filaments

tropomyosin 35 strengthen filaments

myosin light chain 27slide filaments

troponin (T, I, C) 30, 19, 17 mediate regulation of contraction

thymosin 5 sequester actin monomers

extension of the study
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

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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