Proteasome Assay

Before Assay

-Make Lysis Buffer 1, or make sure it is in stock

  • 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

  • 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
  • Working concentration is 40µM LLVY-AMC

Assay

-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)

  • 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 HREXPOSURE: 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

  • Use two 2ml Eppendorf tubes for each condition
  • 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

  • Add 1.5ml of MG132 to one set of wells and 1.5 ml plain media to another set of wells
  • 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)

  • use rubber policeman and scrape cells in each well
  • before scarping the wells from another condition, dip rubber policemen into PBS to wash any cell residues off
  • 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.)
  • 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

  • hide the tube in the ice and place a napkin over the area of the ice where the LLVY tube is hidden
  • 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)

-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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