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Week #5 CHEM 4581 1

Week 5: Electrophoresis

• Collect Homework #3

• This Week in Lab– Lead Cleavage Assay and Urea-PAGE– Qualitative comparison of DNA vs. RNA lanes in

gel

• Lecture: More on Electrophoresis

Week #5 CHEM 4581 2

Gel Matrix Comparison

• Agarose– Polymer of d-galactose + 3,6 anhydro L-

galactose– Use higher conc to separate smaller NA’s– Form Gel via heating and cooling

• Polyacrylamide– Polymer of acrylamide + bisacrylamide– Use higher conc to separate smaller proteins– Form gel via chemical catalysis

Week #5 CHEM 4581 3

Polyacrylamide

Piercenet.com

• [Polyacrylamide] affects pore size

•Small proteins = 30 kD

•Large proteins = 100 kD

• Ratio of A:B affects # of pores

•19:1

•29:1

•37.5:1

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Week #5 CHEM 4581 4

APS & TEMED

• Ammonium persulfate - free radicals• TEMED - stabilizes free radicals

APS

TEMED

Week #5 CHEM 4581 5

Chaotropes and Their Impact

• Potential for H-bonding• “Dehydrates” biological macromolecule• Disrupts aggregates or folding

Chaotropes disruptmacromolecule structure

Week #5 CHEM 4581 6

Sodium Dodecyl Sulfate

SDS confers negative charge on proteins and denatures proteins

Sodium Dodecyl Sulfate(Lauryl Sulfate)

CH3(CH2)11OSO3

Amphiphilic

Hydrophilic Hydrophobic

Anionic Detergentin a wide variety of products

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Week #5 CHEM 4581 7

Buffers maintain pH control Allow gel to fully polymerize

Stacking Gel0.5M Tris-HCl

pH 6.8

Resolving Gel1.5M Tris-HCl

pH 8.8

SDS-PAGE Buffers

Week #5 CHEM 4581 8

Ensure that sample has fully dentured!

Stacking Gel0.5M Tris-HCl

pH 6.8

Resolving Gel1.5M Tris-HCl

pH 8.8

In the Gel In the Sample

Laemmli SampleBuffer

0.5M Tris-HCl,pH 6.8 SDS

Glycerol Bromophenol Blue

Sample Preparation

Boil Sample for 1-5 min

Week #5 CHEM 4581 9

ElectrophoresisBuffer

H3N-CH2-COOH+

AcidicForm

H3N-CH2-COO+ -

ZwitterionicForm

BasicForm

H2N-CH2-COO-

FullyProtonated

Loss of 1Proton

Loss of 2Protons

Tris Base, Glycine, SDS

Electrophoresis Buffer

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Week #5 CHEM 4581 10

Stacking Gel0.5M Tris-HCl

pH 6.8

Resolving Gel1.5M Tris-HCl

pH 8.8

In the Gel

H3N-CH2-COO

H2N-CH2-COO

+ -

Zwitterion Form

-

Basic Form

SDS-PAGE

Week #5 CHEM 4581 11

+

-

Migration in an SDS-PAGE Gel

Week #5 CHEM 4581 12

Migration in an SDS-PAGE Gel

Stop electrophoresis when dye front reaches

bottom of gel

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Week #5 CHEM 4581 13

Coomassie StainCoomassieMethanol

Acetic AciddI Water

Exchangewith

DestainSolution

LeaveOvernight inDestain Soln

Alternatives: Silver StainingFluorescent Dye Staining

Coomassie Staining

Week #5 CHEM 4581 14

log MW

Migration Distance

Analysis of SDS-PAGE Gels

Week #5 CHEM 4581 15

Summary

SDS-PAGE AgaroseProteins, Small DNA/RNA DNA or RNA

Polyacrylamide Agarose

SDS None Req’d

APS, TEMED “Lower Temp”

Tris-HCl TAE, TBE, TPE

Vertical Horizontal

Coomassie Ethidium Bromide

Sample

Matrix

Charge

Catalyst

Buffer

Apparatus

Staining