Separation and Purification of Macromolecules. Why? Molecules each have specific features: Mass Charge Hyrdopathicity.
Post on 26-Mar-2015
222 Views
Preview:
Transcript
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
top related