slide1 l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Comparative Proteomics Kit I: Protein Profiler Module PowerPoint Presentation
Download Presentation
Comparative Proteomics Kit I: Protein Profiler Module

Loading in 2 Seconds...

play fullscreen
1 / 38

Comparative Proteomics Kit I: Protein Profiler Module - PowerPoint PPT Presentation


  • 123 Views
  • Uploaded on

Comparative Proteomics Kit I: Protein Profiler Module. Protein Profiler Kit Instructors. Stan Hitomi Coordinator – Math & Science San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Comparative Proteomics Kit I: Protein Profiler Module' - nam


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
protein profiler kit instructors
Protein Profiler KitInstructors

Stan Hitomi

Coordinator – Math & Science

San Ramon Valley Unified School District

Danville, CA

Kirk Brown

Lead Instructor, Edward Teller Education Center

Science Chair, Tracy High School

and Delta College, Tracy, CA

Sherri Andrews, Ph.D.

Curriculum and Training Specialist

Bio-Rad Laboratories

Essy Levy, M.Sc.

Curriculum and Training Specialist

Bio-Rad Laboratories

why teach protein electrophoresis
Why Teach Protein Electrophoresis?
  • Powerful teaching tool
  • Real-world connections
  • Laboratory extensions
  • Tangible results
  • Link to careers and industry
  • Standards-based
comparative proteomics i protein profiler kit advantages
Comparative Proteomics I: Protein ProfilerKit Advantages
  • Analyze protein profiles from a variety of fish
  • Study protein structure/function
  • Use polyacrylamide electrophoresis to separate proteins by size
  • Construct cladograms using data from students’ gel analysis
  • Compare biochemical and phylogenetic relationships. Hands-onevolution wet lab
  • Sufficient materials for 8 student workstations
  • Can be completed in three 45 minute lab sessions
workshop timeline
WorkshopTimeline
  • Introduction
  • Sample Preparation
  • Load and electrophorese protein samples
  • Compare protein profiles
  • Construct cladograms
  • Stain polyacrylamide gels
  • Laboratory Extensions
traditional systematics and taxonomy
Traditional Systematics and Taxonomy
  • Classification
    • Kingdom
    • Phylum
    • Class
    • Order
    • Family
    • Genus
    • Species
  • Traditional classification based upon traits:
    • Morphological
    • Behavioral
biochemical similarities
Biochemical Similarities
  • Traits are the result of:
    • Structure
    • Function
  • Proteins determine structure and function
  • DNA codes for proteins that confer traits
biochemical differences
Biochemical Differences
  • Changes in DNA lead to proteins with:
    • Different functions
    • Novel traits
    • Positive, negative, or no effects
  • Genetic diversity provides pool for natural selection = evolution
what s in the sample buffer
What’s in theSample Buffer?

• Tris buffer to provide appropriate pH

• SDS (sodium dodecyl sulfate) detergent to dissolve proteins and give them a negative charge

• Glycerol to make samples sink into wells

• Bromophenol Blue dye to visualize samples

why heat the samples
Why Heat the Samples?

• Heating the samples denatures protein complexes, allowing the separation of individual proteins by size

SDS, heat

s-s

Proteins with SDS

+

protein size comparison
Protein Size Comparison
  • Break protein complexes into individual proteins
  • Denature proteins using detergent and heat
  • Separate proteins based on size
protein size
Protein Size
  • Size measured in kilodaltons (kD)
  • Dalton = approximately the mass of one hydrogen atom or 1.66 x 10-24 gram
  • Average amino acid = 110 daltons
actin and myosin
Actin and Myosin
  • Actin
    • 5% of total protein
    • 20% of vertebrate muscle mass
    • 375 amino acids = 42 kD
    • Forms filaments
  • Myosin
    • Tetramer
    • two heavy subunits (220 kD)
    • two light subunits (15-25 kD)
    • Breaks down ATP for muscle contraction
how does an sds page gel work
How Does an SDS-PAGE Gel Work?
  • Negatively charged proteins move to positive electrode
  • Smaller proteins move faster
  • Proteins separate by size

SDS, heat

s-s

Proteins with SDS

+

sds polyacrylamide gel electrophoresis sds page

CH3

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

O

S

O

O

SDS

-

O

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
  • SDS detergent (sodium dodecyl sulfate)
    • Solubilizes and denatures proteins
    • Adds negative charge to proteins
  • Heat denatures proteins
why use polyacrylamide gels to separate proteins
Why Use Polyacrylamide Gels to Separate Proteins?
  • Polyacrylamide gel has a tight matrix
  • Ideal for protein separation
  • Smaller pore size than agarose
  • Proteins much smaller than DNA
    • Average amino acid = 110 daltons
    • Average nucleotide pair = 649 daltons
    • 1 kilobase of DNA = 650 kD
    • 1 kilobase of DNA encodes 333 amino acids = 36 kD
polyacrylamide gel analysis
Polyacrylamide Gel Analysis

Prestained

Standards

Actin & Myosin

Sturgeon

Salmon

Catfish

Shark

Trout

250

150

100

Myosin Heavy Chain (210 kD)

75

50

37

Actin (42 kD)

Tropomyosin (35 kD)

25

20

Myosin Light Chain 1 (21 kD)

15

Myosin Light Chain 2 (19 kD)

10

Myosin Light Chain 3 (16 kD)

can proteins be separated on agarose gels

Prestained

Standards

Actin & Myosin

Prestained

Standards

Actin & Myosin

Sturgeon

Salmon

Catfish

Sturgeon

Shark

Trout

Salmon

Catfish

Shark

Trout

250

150

250

100

Myosin Heavy Chain

Myosin Heavy Chain

150

75

50

100

37

Actin

75

Tropomyosin

50

Actin

25

Tropomyosin

37

20

25

Myosin Light Chains

Myosin Light Chains

15

20

10

Can Proteins be Separated on Agarose Gels?

Polyacrylamide

Agarose

determine size of fish proteins
Determine Size of Fish Proteins

Prestained

Standards

Actin & Myosin

D

A

C

E

B

Measure distance from base of wells to the base of the bands

250

150

100

75

50

37

25

Measure prestained standard bands between ~30 and 10 kD

20

Measure fish protein bands between ~30 and 10 kD

15

10

molecular mass estimation
Molecular Mass Estimation

37 (12 mm)

25 (17 mm)

20 (22 mm)

15 (27.5 mm)

10 (36 mm)

phylogenetic tree
Phylogenetic Tree

Evolutionary tree showing the relationships of eukaryotes.

(Figure adapted from the tree of life web page from

the University of Arizona (www.tolweb.org).)

extensions
Extensions
  • Independent study
  • Western blot analysis
ready gel precast gel assembly
Ready Gel® Precast Gel Assembly

Step 1

Step 2

Step 3

Step 4