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Quality Control Proteins

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X Ray Diffraction. need to crystallize (Difficult) NMR. small proteins 25kD. Electron Micrograph ... X ray diffraction. beam of x rays directed at protein (incident) ... – PowerPoint PPT presentation

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Title: Quality Control Proteins


1
Quality Control Proteins
2
What is Protein QC?
3
What Does Protein QC Entail?
4
QC Protein Analyst Job Description
  • Aerotek Scientific- Boston South, MA
  • Essential Functions
  • Perform GMP Quality Control testing for multiple
    protein-based projects using
  • HPLC
  • ELISA
  • MS
  • Gel
  • Perform data reviews, prepare reports and quality
    documents. Write SOPs.
  • Manage and perform tasks critical for maintaining
    compliance with the applicable regulatory
    requirements.
  • Requirements
  • BS in Biology or Chemistry. 5 years experience
    in Analytical Development and Quality Control for
    protein drugs. Ability to work in GMP and non-GMP
    environments.

5
Why do Protein QC?
6
Who Does Protein QC?
7
Primary Functions
  • Purification
  • Quantification
  • Characterization

8
Purification
9
B I O P R O C E S S I N G
www.integra-biosciences.com
U P S T R E A M
D O W N S T R E A M
10
The Cell
Lyse cells
DNA
Centrifuge Filtration (MWCO)
PROTEIN
11
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12
Chromatography
Tool used to separate a particular protein from
a mixture of proteins and other molecules
13
  • COLUMN
  • MOBILE PHASE
  • PROTEIN SOLUTION
  • STATIONARY PHASE MATRIX (BEADS)
  • ELUTION
  • ELUATE (what flows out)

14
http//www.science.fau.edu/chemistry/Mari/biocheml
ab/05_012.jpg
15
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16
Ion-Exchange Chromatography
  • Separates proteins based on their charge ( or -)

17
Gel-Filtration Chromatography
  • Separates proteins based on SIZE

18
Affinity Chromatography
  • Proteins separates based on ability to bind to
    certain groups
  • based on function/structure

19
Salting Out
  • Precipitation of Proteins in Solution
  • Ammonium Sulfate
  • Mechanism
  • Proteins have hydration layer (H2O-polar side
    groups)
  • If this layer is disrupted, solubility decreases
  • Ions (NH4, SO42-) disrupt hydration layer
  • because the ions what to be hydrated
  • Gradient some protein precipitate, some dont

20
Electrophoresis
  • Separate proteins based on
  • Size (Molecular Weight - MW)
  • SDS PAGE
  • Isoelectric Point
  • Isoelectric focusing

21
Allows us to
  • characterize (degradation, MW)
  • quantify
  • determine purity of sample
  • compare proteins from different sources
  • step in Western blot

22
SDS-PAGE
Sodium Dodecyl Sulfate - Polyacrylamide Gel
Electrophoresis developed by Laemmli
(1970) Denatured Gel
23
How to Detect Proteins?
Coomassie Blue (0.1 ug) Silver Staining (2 ng)
How to Quantify ProteinsDensitometry
24
Molecular Weight Determination
Method 1 Amino Acids approx 110 daltons
residues x 110 dalton/residue MW Method
2 Run SDS PAGE with known standards (MW
markers) Graph Measure distance unknown protein
traveled Compare on standard curve
25
Immunoblots (Westerns)
26
Quantification
27
Antibodies as Reagents
Immunoassays - assays which use Ab to detect and
quantify substances Ab are extremely specific -
ADVANTAGE Ab can not be detected, need a
marker Radioactive labels (RIA) Enzymes (EIA
) FIA
28
Enzyme Immunoassay
ELISA (antibody sandwich) Enzymes used
horseradish peroxidase, alkaline
phosphatase Alternates Fluorescent
tag Chemiliminescent tags
29
http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/E/ELISA.gif
30
Spectroscopy
31
  • based on an absorbance shift in the dye Coomassie
  • when bound to arginine and hydrophobic amino acid
    residues present in protein
  • Higher the A595 Higher the amount of protein

32
Absorption Transmission Reflection Leaves
Reflect 500-600 nm- This wavelength is not
absorbed by chlorophyll What color is this?
33
A280
Tryphophan Phenylalanine Tyrosine ALL ABSORB
LIGHT AT 280 nm Crude, not necessarily
quantitative Same amount of protein will show
different A280 depending on amount of above amino
acids UV IR
34
Bradford Assay
SECOND MOST CITED PAPER IN SCIENCE
JOURNALS Bradford, M. M. (1976) A Rapid and
Sensitive Method for the Quantitation of
Microgram Quantities of Protein Utilizing the
Principle of Protein-Dye Binding. Anal.
Biochem. 72248-254.
Coomassie Brilliant Blue G Dye
35
Characterization
36
Proteins Biochemistry and Biotechnology, Walsh,
Wiley Press
37
Amino Acid Sequencing
  • Edman Degradation

http//www.unipa.it/cobs/en/protmic/standard.gif
38
Amino Acid Composition
Treat with HCl (hydrolysis) Separate individual
aa by ion exchange chromatography Analyse with
HPLC
39
3D Structure Determination
  • X Ray Diffraction
  • need to crystallize (Difficult)
  • NMR
  • small proteins 25kD
  • Electron Micrograph
  • poor resolution

40
X-ray Crystallography
  • X ray diffraction
  • beam of x rays directed at protein (incident)
  • beam is diffracted by electrons of atoms in
    protein (scattered)
  • these beams hit a film detector
  • computer analysis to create electron density map

41
NMR Nuclear Magnetic Resonance
  • Use radio frequency pulses
  • Energy is absorbed such that move from ground to
    excited states
  • atomic nuclei spin - create their own magnetic
    field
  • emit radiation differs based on atoms or
    chemical groups

42
What is Spin?
  • Describes magnetic field around a nucleus
  • No magnetic Filed, Spin 0
  • N P
  • When N P Odd number, Spin 1/2

43
Electron Microscopy
Creating image by using electrons rather than
visible light Particularly for objects having
dimensions smaller than the wavelengths of
visible light
44
Electron Microscopy
  • Direct e- through sample
  • When e- hit nuclei or electrons they scatter
  • Capture electrons
  • Transmissiom EM, Scanning EM

45
  • Transmission (TEM)
  • Scanning (SEM)

Nobelprize.org
www.mos.org
46
Mass Spectrometry
Molecular Mass determination More accurate than
SDS PAGE Less protein needed for analysis than
SDS PAGE
http//www.chem.arizona.edu
47
Mass Spectrophotometry
the mass of the ion. Lighter ions are deflected
more than heavier ones.
48
Mass Spec Analysis
(mass to charge ratio)
http//chipo.chem.uic.edu/web1/ocol/spec/MS.htm
49
HPLC
  • Used to characterize
  • ID
  • Quantify
  • Purify

50
HPLC
  • Column Selection
  • Solvent Selection
  • Gradient vs isocratic runs
  • Sample pre
  • Standards (labeled unlabeled)

51
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52
  • fig.cox.miami.edu
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