Proteasome Assay
Before Assay
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Make Lysis Buffer 1, or make sure it is in stock
o 10mM Tris-HCl, 0.5mM DDT, 5mM ATP, 0.035% SDS, 5mM MgCl2,
pH 7.8
 Add 788mg Tris-HCl, 175mg SDS, and 508.25mg MgCl2 into 500 ml
of milli Q water
 The concentrations of ATP and DDT have to be calculated depending
on the amount of lysis buffer that is used for the experiment
 NOTE: ATP and DTT are added to the lysis buffer right before the
lysis buffer is added to the cells
Dilute LLVY-MCA to 4mM, aliquot, and store in -20˚C tissue culture freezer in
the dark
o 5mg LLVY-MCA + 163.6μl DMSO = 40mM
 Aliquot 5.0μl LLVY-MCA into 0.5ml Eppendorf tubes and store in 20˚C tissue culture freezer in the dark
o Working concentration is 40µM LLVY-AMC
Assay
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Use 6-well plates for this experiment
Use one plate for each condition to assure that only the cells that are treated at
given time points are removed from the incubator
expose cells (HT-22 and CHIP-myc) to several different conditions (0hr, 0hr +
MG132, 6hr + 3mM Glutamate, 6hr + MG132)
o add 1.5ml of respective solution into the respective wells
start the condition that has the longest time frame (i.e., add the 3mM glutamate
and the MG132 to the cells that are going to be exposed for 6hrs first. Then add
the glutamate and MG132 to the wells of decreasing exposure periods.)
IMPORTANT NOTE FOR 0 HR EXPOSURE: 30 min before the overall time
(i.e. longest time point, which in this case would be 6hrs) is over, replace the
media in the 0hr control wells with 1.5ml plain media (HT-22 and HT-22 + Hygro
media in appropriate wells), and incubate 2 wells of the naïve cells in the 6-well
plate with 50μg MG132 for 30 min
After the cells for the longest time period have been treated, 2ml and 0.5ml
Eppendorf tubes have to be labeled
o Use two 2ml Eppendorf tubes for each condition
o Use one 0.5 ml Eppendorf tube for each condition (this tube will contain a
small sample for the protein assay)
30 minutes before the overall time is up, the 0hr time point cells have to be treated
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o Add 1.5ml of MG132 to one set of wells and 1.5 ml plain media to another
set of wells
o Place plates back into the incubator
get a bucket of ice
add 3.5ml of lysis buffer into an empty 15 ml tube and place on ice
fill up a small plastic beaker with PBS and place rubber policeman into it
when time is up, harvest all cells of the same type first, and then all cells of the
other condition (e.g., harvest all HT-22 cells first, and then all CHIP-myc cells)
o use rubber policeman and scrape cells in each well
o before scarping the wells from another condition, dip rubber policemen
into PBS to wash any cell residues off
o once all cells from one cell type are scarped off, transfer media into
labeled, 2ml Epi tubes and PLACE ON ICE!!!
 for each condition, there are two 2ml Epi tubes
 add 1.5 ml of each condition into each of the two Epi tubes (e.g, for
HT-22 0hr, there is a total of 3ml in the two wells of the 6-well plate.
After scarping the cells of the bottom of the wells, place 1.5 ml of
media with cells into one epi tube and 1.5 ml of media into the other
epi tube.)
o after all the plates from one cell type are harvested, repeat the same steps
for all the other cell types, until all cells are harvestes
spin cells down (10 min at 2rpm) in centrifuge in cold room
while cells are spinning add 175μl ATP and 3.5μl DTT into lysis buffer, shake the
solution a little bit and place on ice
get LLVY out of freezer
o hide the tube in the ice and place a napkin over the area of the ice where
the LLVY tube is hidden
o LLVY has to be protected from the light, since it is light sensitive
after the cells are centrifuged, remove the media carefully with a glass pipette,
and this time work with ONE condition at a time (i.e., take care of HT-22 0hr
first, and then HT-22 0hr + Mg 132, and so forth)
after the media has been removed, wash pellet with cold 1x PBS (2x) and quickly
add 400μl lysis buffer, and resuspend cell by pipetting the buffer up and down.
Suck up all the buffer from the first tube and place it into the second tube
Pipette up and down to resuspend the cells in the second tube
Add 50μl into a respective small tube and place on ice
Place the tube with the remaining 350μl on ice as well
Repeat steps with all conditions
When all conditions are done add 0.35μl LLVY into the tubes that contain the
350μl lysis buffer
Place tubes in water bath (37ºC) for 30 min
Bring the small tubes for the protein assay to the freezer and store them there until
they are needed for the protein assay
Get a WHITE 96 well plate ready (bring it to the cell culture room and have it
ready to be loaded with samples)
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Once the 30min are up, take tubes out of water bath and add 87.5μl tissue culture
H20 and 13.125μl ethanol to each tube
Shake the tubes and add 100μl of each sample into four wells on plate
Put plate into the plate reader with parameters set for fluorescence at 360 nm
excitation and 465nm emission
IMPORTANT NOTE: The values for ATP, DTT, H20 and Ethanol have to be
calculated according to the amount of lysis buffer that has been used!
Jeannette Stankowski
01.08.07
2.28.07
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