Separation and Purification of Macromolecules. Why? Molecules each have specific features: Mass Charge Hyrdopathicity.

Separation and Purification of Macromolecules

Why?

Molecules each have specific features:

Mass Charge Hyrdopathicity

How?

Gel electrophoresis

Chromatography

Gel electrophoresisGel electrophoresis

1.1. Typically used for separation Typically used for separation by mass.by mass.

2. Molecule is forced through a 2. Molecule is forced through a physical matrix. physical matrix.

MatrixMatrix

Smaller molecules migrate further.Smaller molecules migrate further.

Uses electric field to “move” molecules.Uses electric field to “move” molecules.

What is DNA’s charge???What is DNA’s charge???

Agarose GelsAgarose Gels

Typically used for Typically used for DNADNA

Can accurately move Can accurately move 150bp and above150bp and above

SDS-PAGESDS-PAGE

Poly Acrylamide Gel ElectrophoresisPoly Acrylamide Gel Electrophoresis

Smaller openings in matrixSmaller openings in matrix Used for smaller macromoleculesUsed for smaller macromolecules ProteinsProteins

Why use SDS???Why use SDS??? SDS adds a charge that is proportional to the mass.SDS adds a charge that is proportional to the mass.

2D Gels2D Gels

2 Step process2 Step process

Separate by Separate by Isoelectric Isoelectric point (pH)point (pH)

Then separate Then separate by SDS-Pageby SDS-Page

ChromatographyChromatography

Used to separate by several Used to separate by several characteristics characteristics Mass, charge, hydropathicityMass, charge, hydropathicity

Two phasesTwo phases Mobile phase (typically liquid)Mobile phase (typically liquid) Solid phase (typically solid)Solid phase (typically solid)

chromatography paperchromatography paper

Separation occurs based on attraction to Separation occurs based on attraction to one phase over the other.one phase over the other.

Chromatography Chromatography ExamplesExamples

HydropathicityHydropathicity Polar/nonpolar phasesPolar/nonpolar phases

ChargeCharge Neutral solvent/ charged matrixNeutral solvent/ charged matrix

RfRf

=(Distance band moved / distance =(Distance band moved / distance solvent moved)solvent moved)

Need to know this for the homeworkNeed to know this for the homework

T-testT-test

Used to determine if two sets of data can Used to determine if two sets of data can be explained by randomness.be explained by randomness. You want to make sure your information is You want to make sure your information is

not random.not random.

T-test SignificanceT-test Significance

Indicates “real” differences in valuesIndicates “real” differences in values Used to distinguish significance of difference Used to distinguish significance of difference

between experimental and control sets.between experimental and control sets. Given in p valueGiven in p value

P=probability that differences are randomP=probability that differences are random (1.00-p) is indicator of significance(1.00-p) is indicator of significance Typically p<0.05 is cutoff point (0.95 significance)Typically p<0.05 is cutoff point (0.95 significance)

5% chance that differences occurred randomly5% chance that differences occurred randomly