Isolation of biological macromolecule

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Isolation of biological macromolecule
Technology to simply go into a mixture and grab a
single type of molecule is not readily
available Instead use procedures to eliminate or
exclude other types of molecules leaving the
desired one behind Achieved by taking
advantages of differences between molecules
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Example Plasmid prep
Need to eliminate membranes, proteins,
chromosomal DNA, and RNA Lyse cells (rupture
membranes) typically with alkaline lysis High pH
and detergents will lyse cells Then neutralize
pH which causes membranes to clump Chromosomal
DNA stays attached to membranes unless it is
sheared by rough pipetting or
vortexing Centrifuge to pellet
membrane/chromosome complex Alkaline lysis
solutions also contain Rnase which degrade RNA
rapidly Left with proteins and plasmid DNA in
solution Load on spin column with nylon membrane
DNA will bind to nylon presence of alcohol
strengthens this proteins will spin through
membrane in the presence of alcohol After this
step, only plasmid DNA should be left on
membrane add water or buffer, let plasmid DNA
leave membrane and spin through into fresh
tube
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Variation on conceptual theme
Same idea could play out with different
steps Boiling miniprep of plasmid DNA Cells are
lysed by putting cells with lysozyme, then
putting in boiling water remove membranes and
chromosomes by centrifugation RNA is degraded by
Rnase Proteins are removed by phenol
extraction phenol is non-polar so proteins will
tend to collect at polar/nonpolar interface of
extraction Collect aqueous phase which should
have plasmid DNA Precipitate plasmid DNA DNA
will precipitate in cold ethanol with NaCl
present Pellet precipitated plasmid DNA by
centrifugation Dry and resuspend pellet in
water or buffer Same general concept of
sequential exclusion, but specific mechanisms are
different
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Isolating chromosomal DNA
Lyse cells with detergent at neutral pH and
chromosome will not adhere to membranes Degrade
RNA with Rnase Remove proteins with proteinase
and/or by phenol extraction Chromosomal DNA will
make long strands when precipitated in
isopropanol plasmids will not make
strands Hook stranded DNA form precipitation and
resuspend
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Isolating RNA
Lyse cells and remove membranes by
centrifugation Degrade DNA with Dnase
enzyme Remove proteins by protease and/or phenol
extraction Precipitate RNA in very cold ethanol
precipitation or on column
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Isolating Proteins
Lyse cells and remove membranes by
centrifugation Degrade RNA with Rnase Degrade
DNA with Dnase Precipitate protein with ammonium
sulfate Suspend and dialyze to remove excess salt