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

ifferent methods have different levels of

detection ie, values can go from grams to
nanograms
Electrophoresis and
!ovement of !olecules

!olecules can have distinct charges

Positive or "egative

"et charge #ill cause different movement

through gel

!olecules can have different shapes

$inear

globular

Alpha helix
%
!acromolecular charge

!acromolecules have a
variable net charge that
depends on p&

p& at #hich net charge is

'ero ( p)

Electrical shielding of
charge occurs #hen
counterions are solvated
*(
* (
Electrophoresis

&ori'ontal Agarose Gels

Agarose forms a gel or molecular sieve

that supports the movement of small
materials in solution used for "A
+
*ertical Polyacrylamide Gels

!ade of Polyacrylamide

,sed for Protein molecular si'e, shape,

charge

)E- electrophoresis

.estern /lot techni0ue

&ori'ontal Gels

Gel /ox set up fre0uently used in "A

analysis
Agarose gels

,sually used in "A analysis

!ade up of linear polysaccharide mol #t of 12,333

/asic repeating unit is agarobiose

Gels are prepared at 14 to 54 providing tunnels for

molecules to move through

"A can be much larger then most proteins

Agarose Gel #ith "A /ands

"A is negatively
charged

6maller si'ed "A

moves faster than
$arger "A

!ar7ers are used to

determine relative
si'es of "A pieces
mar7ers
PAGE

"ative 8 Protein is prepared #ith little

disturbance to the cellular material

Proteins are associated

!ovement of samples through the gel can be

inconsistent

66 8 6odium odecyl 6ulfate )s a

detergent

Protein coated #ith a negative charge in

proportion to its molecular #eight

enatures and unfolds protein

9educing agents :TT;brea7 amino acid

cross<lin7s
P
Polyacrylamide Gel
Creates tunnels in gel for molecules to move
through
,ses for PAGE

6eparates proteins from each other

Proteins separated by si'e

)soelectric point

etermines

!olecular si'e of protein

Quantifies the amount present

isplays )mpurities

,sed in #estern blot assays by antigen

interactions
etermine !olecular .eight
1= 9un standard molecular #eight mar7ers
on gel
2= 9un un7no#n protein on the same gel
5= Create a graph of the mol #t versus
distance molecule has moved
>= ,sing the distance the un7no#n has
moved determine the molecular #eight from
graph
!olecular .eight !ar7ers
!igration of molecular #eight
of standards are compared to
un7no#n sample#t std vs
un7no#n
!olecular .eight vs istance
.estern /lot Analysis

)dentifies protein through antibody

interaction

9un proteins on denatured gel :66<PAGE;

Transfer :blot; proteins onto membrane

Probe the membrane #ith primary antibody

Add secondary antibody :this antibody is

lin7ed to an en'yme;

6ubstrate is added and color appears

66 Polyacrylamide
Electrophoresis
66 Effect on Protein !ovement

6odium odecyl 6ulfate denatures protein

and covers it #ith negative charges 8
moves to % end

*ertical gels are designed so the top of the

gel box is attached to the negative po#er
outlet

The bottom of the gel box is attached to the

positive po#er outlet

!ovement through the PAGE gel is

proportional to mass not to charge
!ovement of Proteins on an
66 Gel
6tac7ing of
proteins at top
of gel at start
$o# #eight
molecular dye
<
%
istribution of
proteins in a
charged field
Protein !igration
&ighest
!olecular
.t= protein
4 Polyacrylamide in Gel

Gels can be made at different

concentrations of polyacrylamide

Example8 gels made at 54,?4,@4 and 124

#ill produce different openings through
#hich the molecule #ill migrate

The larger the opening allo#s large

molecules to move through the gel
*ertical Polyacrylamide Gel
Electrophoresis
E0uipment for Electrophoresis
Gel Electrophoresis E0uipment
!ini<P9ATEA" Tetra Cell
Closed !ini Gel holder
Apen Gel &older8
Allo#s "e# Gel to be )nserted
Gel &olders
Placed in !ini<Protean Tetra Cell
Procedure in 6hort
$oadGe Place /uffer
E0uip
Electrophoresis of 6amples
6etting
,p and 9unning !ini<P9ATEA" TGB Precast Gels
C
Douhttp
8EE###=youtube=comE#atchFv(BnEdm7160vgTube
6amples8 boiled 5G #ith loading
dye :2x $aemmli buffer %
running dye;
!ini<P9ATEA" tetra cell8 6et
up according to 6AP given in
#or7boo7
Po#er settings8 HI volts for >I
C ?3 minutes
9unning dye should not
run off the bottom of gel