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

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Presentation transcript:

Separation and Purification of Macromolecules

Why? Molecules each have specific features: Mass Charge Hyrdopathicity

How? Gel electrophoresis Chromatography

Gel electrophoresis 1.Typically used for separation by mass. 2. Molecule is forced through a physical matrix.

Matrix Smaller molecules migrate further. Smaller molecules migrate further. Uses electric field to move molecules. Uses electric field to move molecules. What is DNAs charge??? What is DNAs charge???

Agarose Gels Typically used for DNA Typically used for DNA Can accurately move 150bp and above Can accurately move 150bp and above

SDS-PAGE Poly Acrylamide Gel Electrophoresis Poly Acrylamide Gel Electrophoresis Smaller openings in matrix Smaller openings in matrix Used for smaller macromolecules Used for smaller macromolecules Proteins Proteins 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 Gels 2 Step process 2 Step process Separate by Isoelectric point (pH) Separate by Isoelectric point (pH) Then separate by SDS-Page Then separate by SDS-Page

Chromatography Used to separate by several characteristics Used to separate by several characteristics Mass, charge, hydropathicity Mass, charge, hydropathicity Two phases Two phases Mobile phase (typically liquid) Mobile phase (typically liquid) Solid phase (typically solid) Solid phase (typically solid) chromatography paper chromatography paper Separation occurs based on attraction to one phase over the other. Separation occurs based on attraction to one phase over the other.

Chromatography Examples Hydropathicity Hydropathicity Polar/nonpolar phases Polar/nonpolar phases Charge Charge Neutral solvent/ charged matrix Neutral solvent/ charged matrix

Rf =(Distance band moved / distance solvent moved) =(Distance band moved / distance solvent moved) Need to know this for the homework Need to know this for the homework

T-test Used to determine if two sets of data can be explained by randomness. Used to determine if two sets of data can be explained by randomness. You want to make sure your information is not random. You want to make sure your information is not random.

T-test Significance Indicates real differences in values Indicates real differences in values Used to distinguish significance of difference between experimental and control sets. Used to distinguish significance of difference between experimental and control sets. Given in p value Given in p value P=probability that differences are random P=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 randomly 5% chance that differences occurred randomly