Transcript Document

Quality Control of Product
Polyacrylamide Gel Electrophoresis
Analysis of Product
• Quality Control involves the entire process of
obtaining a product that meets defined
specifications expressing both its purity and
potency
• Testing methods include cell biology, virology,
chemistry, analytical chemistry, molecular
biology & the potency of the product
• Different methods have different levels of
detection ie, values can go from grams to
nanograms
Electrophoresis and
Movement of Molecules
• Molecules can have distinct charges
– Positive or Negative
– Net charge will cause different movement through
+
gel
• Molecules can have different shapes
– Linear
– globular
– Alpha helix
Macromolecular charge
• Macromolecules have a
variable net charge that
depends on pH
• pH at which net charge is
zero = pI
• Electrical shielding of charge
occurs when counterions
are solvated
V=
V=
Electrophoresis
• Horizontal Agarose Gels
• Agarose forms a gel or molecular sieve
that supports the movement of small
materials in solution used for DNA
• Vertical Polyacrylamide Gels
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Made of Polyacrylamide
Used for Protein molecular size, shape, charge
IEF electrophoresis
Western Blot technique
Horizontal Gels
• Gel Box set up frequently used in DNA analysis
Agarose gels
• Usually used in DNA analysis
• Made up of linear polysaccharide mol wt of
12,000
• Basic repeating unit is agarobiose
• Gels are prepared at 1% to 3% providing
tunnels for molecules to move through
• DNA can be much larger then most proteins
Agarose Gel with DNA Bands
markers
• DNA is negatively
charged
• Smaller sized DNA
moves faster than
Larger DNA
• Markers are used to
determine relative
sizes of DNA pieces
PAGE
• Native : Protein is prepared with little
disturbance to the cellular material
– Proteins are associated
– Movement of samples through the gel can be
inconsistent
• SDS : Sodium Dodecyl Sulfate Is a detergent
– Protein coated with a negative charge in
proportion to its molecular weight
– Denatures and unfolds protein
– Reducing agents (DTT)break amino acid cross-links
Polyacrylamide Gel
P
Creates tunnels in gel for molecules to move through
Uses for PAGE
• Separates proteins from each other
– Proteins separated by size
– Isoelectric point
• Determines
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Molecular size of protein
Quantifies the amount present
Displays Impurities
Used in western blot assays by antigen interactions
Determine Molecular Weight
1. Run standard molecular weight markers
on gel
2. Run unknown protein on the same gel
3. Create a graph of the mol wt versus
distance molecule has moved
4. Using the distance the unknown has moved
determine the molecular weight from graph
Molecular Weight Markers
Migration of molecular weight
of standards are compared to
unknown samplewt std vs
unknown
Molecular Weight vs Distance
Western Blot Analysis
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Identifies protein through antibody interaction
Run proteins on denatured gel (SDS-PAGE)
Transfer (blot) proteins onto membrane
Probe the membrane with primary antibody
Add secondary antibody (this antibody is linked
to an enzyme)
• Substrate is added and color appears
SDS Polyacrylamide Electrophoresis
SDS Effect on Protein Movement
• Sodium Dodecyl Sulfate denatures protein and
covers it with negative charges : moves to + end
• Vertical gels are designed so the top of the gel
box is attached to the negative power outlet
• The bottom of the gel box is attached to the
positive power outlet
• Movement through the PAGE gel is
proportional to mass not to charge
Movement of Proteins on an SDS
Gel
Protein Migration
Stacking of
proteins at top of
gel at start
Highest
Molecular
Wt. protein
Distribution of
proteins in a
charged field
+
Low weight
molecular dye
% Polyacrylamide in Gel
• Gels can be made at different concentrations of
polyacrylamide
• Example: gels made at 3%,6%,9% and 12% will
produce different openings through which the
molecule will migrate
• The larger the opening allows large molecules
to move through the gel
Vertical Polyacrylamide Gel
Electrophoresis
Equipment for Electrophoresis
Gel Electrophoresis Equipment
Mini-PROTEAN Tetra Cell
Closed Mini Gel holder
Open Gel Holder:
Allows New Gel to be Inserted
Gel Holders
Placed in Mini-Protean Tetra Cell
Procedure in Short
LoadGe
Equip
Place Buffer
Electrophoresis of Samples
Setting Up and Running
Samples: boiled 3’ with
Mini-PROTEAN TGX Precast loading dye (2x Laemmli
Gels –
buffer + running dye)
Mini-PROTEAN tetra cell:
Youhttp://www.youtube.co Set up according to SOP
m/watch?v=XnEdmk1SqvgT given in workbook
ube
Power settings: 75 volts for
45 – 60 minutes
Running dye should not
run off the bottom of gel