ANAT 262 Lecture Notes - Column Chromatography, Cytosol, Electrophoresis

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Published on 21 Aug 2012
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In Vitro Systems:
In vitro cell-free system: used to study a particular aspect of a process which is re-
duced to its minimal components (bottom up way to look at the cell)
-ideally, looking at only one process and you know every ingredient that goes in
but usually you are purifying a whole organelle starting with cytosol and trying to
reduce it to a few components that you do understand
-use an assay to determine whether and how efficiently the desired process took place
-usually start with uncharacterized cytosol for the initial development of an in vitro sys-
tem and then replace this with purified proteins to allow determination of the minimal
proteins required for the process to occur
example: translocation into ER needs only purified microsomes, cytosol and mRNA
(these must be the requirements for transport such as ATP, GTP, and particular proteins)
-purify ER membranes - get mix of rough and smooth microsomes (only interested in
rough ribosomes = protein synthesis)
-suspend, load onto gradient, and then rough microsomes go to the bottom
-use EM to see what the fractions were...next added proteases which will chop
up anything produced outside the lumen (proteins become protease resistant if
they get into the lumen)...
-add mRNA that codes for antibody light channels and use in vitro systems to
eliminates some of the components
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-without microsomes or protease it makes a light chain in SDS gel electrophoresis
-if you add protease without microsomes it knocks out the protein all together (gets rid
of light chain - if you are not protected the protease knocks you out)
-if you add microsomes but no protease you get a light chain but it is farther down
meaning it is lighter...so a piece is chopped off (signal sequence - which is required to
get into the ER)
-if you add both microsomes and protease the top of the band disappears and you just
have one band...
process: translocation into the ER
-assay: proteins become protease resistant if they get into the ER, molecular
weight changes during the translocation process (piece is chopped off the pro-
tein when translocated)
Density Gradient Centrifugation: take cell and break open to get ER into microsomes
(small balls separated into rough or smooth ER) - need to use a tube with gradient of in-
creasing density as you go towards the bottom of the fluid
-once spun with a centrifuge the denser microsomes (those with the ribosomes -- rough
ER) will sink to the bottom (heavier) and the smooth ribosomes will be on top (lighter)
-protein synthesis occurs in ribosomes (translocation)
-once proteins are inside the lumen they are protease resistant
Protein Separation Technique - gel filtration chromatography - the principle of sep-
aration is based on volumes
-have a large column which is full of beads that have holes in them, then add liquid to
top which will be pushed to the bottom by gravity
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-large molecules cannot get into the beads at all, medium molecules will mostly
stay outside the beads although a few may get in, and small proteins will make it
into the beads
-so the large you are the faster you go through the column
-can be used with large amounts of proteins to purify them
Gel Electrophoresis - load proteins into top of the gel, and allow proteins to move
in the electric field (negative at top and positive at bottom)
SDS-PAGE: separate based on size of the proteins - load proteins at the top of the
gel -- proteins will separate based on size (large proteins go through slowly)
-problem: proteins have different shapes
-solution: add SDS to denature the proteins so they will all be the same rod
shape (can also add reducing agent to break sulfide bonds)
-more charge from SDS than from the charge from the proteins (all proteins will
be rods with the same amount of charge per unit length of rod -- proteins will be
separated only by size)
-if proteins are glycoproteins the molecular weights will be off (but for other pro-
teins you can determine the molecular weights and thus see what proteins are
in a sample)
-if you add radioactive proteins you can visualize the proteins
For an In vitro system you need:
1) sufficient components to support the process of interest - may need purified mole-
cules, organelles, or partially intact cells
2) a process that the investigator wants to study
3) an assay to determine whether and how efficiently the desired process took place
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Document Summary

In vitro cell-free system: used to study a particular aspect of a process which is re- duced to its minimal components (bottom up way to look at the cell) Ideally, looking at only one process and you know every ingredient that goes in but usually you are purifying a whole organelle starting with cytosol and trying to reduce it to a few components that you do understand. Use an assay to determine whether and how efficiently the desired process took place. Purify er membranes - get mix of rough and smooth microsomes (only interested in rough ribosomes = protein synthesis) Suspend, load onto gradient, and then rough microsomes go to the bottom. Use em to see what the fractions werenext added proteases which will chop up anything produced outside the lumen (proteins become protease resistant if they get into the lumen) Add mrna that codes for antibody light channels and use in vitro systems to eliminates some of the components.

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