Ellyns Top 10 Protein Labs Part 1

1
Ellyns Top 10 Protein Labs(Part 1)

• Ellyn Daugherty
• SM Biotech Career Pathway
• www.SMBiotech.com
• www.BiotechEd.com
• www.emcp.com/biotech
• www.sargentwelch.com/biotech
• AEEDaugher_at_aol.com
• 650-400-9424

2
What are Proteins? Why are they so Important?

• Functions
• Hormones, antibodies, pigments,
• transport, contractile, structural,
• recognition, and enzymes
• Structure
• Large macromolecules (AA necklaces)
• Diff s and combos of 20 diff AA
• Important because
• There are so many different proteins with so many
  functions.
• Over 2000 different proteins/cell (humans make
  over 40K)
• So complex and sensitive to changes-cause big
  change
• Often the product of a biotech company!
• Foods/Environmental,Pharmaceuticals/Drug
  Development, Industrial Products

Sickled cell due to faulty protein
3
Examples of Protein Function

4
Why Study Proteins?

• To increase scientific knowledge (basic science)
• gt understand processes
• gt biochemical pathways
• gt evolutionary studies
• To make a (protein) product.
• gt Herceptin (breast cancer antibody)

We must understand structure and function to
produce, purify, and quantify proteins during
manufacturing
5
To make a (protein) product. Protein Production
at a Typical Biotech Co.

• Cells are genetically engineered to make new
  proteins.
• Engineered cells are scaled up to progressively
  large volumes, producing larger amounts of
  protein.
• Protein must be assayed.
• Protein must be purified.
• Protein must be formulated.

6
I like to say

• DNA is the Flash
• but
• Proteins are the Cash of Biotech

7
Lab 1c Cheese-Making

• Uses real proteins (the enzymes, rennin and
  chymosin) used in industry and shows how biotech
  has impacted an industry.
• In addition
• Good for introducing scientific methodology
  (experimental design, data collection/analysis,
  conclusions) and introducing bioethics.
• Demonstrates the effects of too many variables in
  an experiment.
• Good for introducing quantification and
  measurement, as with the whey-o-meter, pipets and
  micropipets.
• Do Lab 2f as follow-up on how pH affects protein
  structure.

8
Lab 5a Rockys Allergies

• Ouchterlony Testing Simulation
• (One of the few simulations I do.)
• Good way to introduce Ab, Ag before using the
  expensive real stuff.
• Fast, easy, and very visual.
• Follow it up with Lab 14a Pig Contamination ELISA

9
Lab 6c Amylase Assay

• Amylase converts substrate (starch) to product
  (maltose)
• Can measure substrate decrease or product increase

10
Lab 5f and 5gSeparating Proteins via SDS-PAGE
Gels (Polyacyrlamide Gel Electrophoresis)
11
PAGE (denaturing/sizing) Gels

• Lab 5f Protein/Peptide Size Characterization
• of chains in the protein
• Size (kD) chain and approximate AA
• Size (kD) protein and approximate AA

• Lab 5g Muscle Tissue Protein Comparison
• Extract proteins from cells
• Look for similarities and differences in protein
  content
• Relate protein composition to tissue function

12
6f, 6g, 6h Peroxidase Labs

• Peroxidase Assay (Lab 6f)
• HRP extraction from HR Root (Lab 6g)
• HRP assay using PAGE (lab 6g)
• HRP assay w/ TMB indicator (Lab 6h)
• HRP used in ELISA (Lab 14a)

13
And my 1 The Spec Labsall of Chapter 7
14
Lab 7fAbsorbance Spectrum of a Protein Molecule

• Lab 7b and 7f Determine the wavelength where the
  molecule absorbs the most light.
• Lambda max
• Lab 7b and 7f
• Use Lambda max to visualize protein
• Lab 7f - Use a protein indicator (Bradford
  reagent) to make protein (amylase) visible in spec

15
Lab 7g Determining Protein Concentration

• Standards show Concentration vs Abs Relationship
• Use protein solutions of known concentration to
  estimate unknowns.

• y mxb
• where y the absorbance of the unknown sample
• m the slope of the best-fit straight line
• x the concentration of the unknown sample
• b the y-intercept

16
Biotechnology Science for the New Millennium
Ellyn Daugherty www.emcp.com/biotech

• Text with Encore CD
• Lab Manual
• Instructors Guide
• Course Planner (Lesson Plans)
• Instructor Resource Center on web
• Student Notebook, Test Generator, etc. available

www.BiotechEd.com for tour through the entire
curriculum, ancillaries, and websites