Team:Freiburg/Labbook/Proteasome
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23.09.19
Mixed lysis buffer:
50 mM HEPES (pH 7.8)
10 mM NaCl
1.5 mM MgCl2
1 mM EDTA
1 mM EGTA
250 mM sucrose
Sterile filtration
Added DTT to 5 mM final concentration (used 1 M stock)
Mixed assay buffer:
50 mM HEPES (pH 7.8)
10 mM NaCl
1.5 mM MgCl2
1 mM EDTA
1 mM EGTA
250 mM sucrose
Sterile filtration.
Added DTT to 5 mM final concentration (used 1 M stock) and ATP to a final concentration of 2mM.
Seeded equal amounts of HEK293 cells into 5 wells of a cell-culture treated 6-well plate in 1.6 mL RPMI medium with 10% FCS and 1mM HEPES.
Incubated for approx. 20h.
24.09.19
Fresh RPMI without anything for all cells. Added 100 μM of D and L-PSMα3 to two wells, nothing to the other 3, incubated 1 h.
Washed 1x with icecold PBS, resuspended cells in 400 μL lysis buffer, dissolved cells from culture dish by pipetting up and down. Kept on ice afterwards.
The lysates of equally treated wells were pooled.
Sonicated for 3* 30 s (30 s break)
Centrifuged 10 min 16.000 g at 4°C.
Transfered supernatant into 5 centrifuge tubes: water control, cells treated with L / D PSMα3, lysates L and D.
(Lysates L and D were mixed with 100 μM L and D-PSMα3respectively).
Transferred 50 μL of lysate into black 96 well plate and added 200 μL of assay buffer. Preincubation at 37°C for 10-15 min.
Made 5 mM stock of Z-LLL-AMC fluorogenic substrate in 50% DMSO 50% nuclease free water.
Added 5 μL of substrate to each well of the black plate ( final concentration of 100 μM).
Incubated 60 min at 37°C.
Measured fluorescence at A360ex/A460em in a plate reader.
25.09.19
Seeded HEK293 cells into 6 wells of a cell culture treated 6-well plate.
26.09.2019
When HEK293 had confluency 70%, washed carfully with PBS, added 400 uL lysis buffer, resuspended and sonicated cells (2x 30s).
Centrifuged lysates at 16000g at 4°C for 10 min.
Pooled the lysates and added 25 uL to each well of a black 96 well plate, added substances according to the layout below:
| PSMa3 (20uM) | LLL-AMC | ATP | preincubation of PSMa3 with lysates, without LLL-AMC |
| - | + | + | - |
| - | + | + | - |
| L-PSMa3 | + | + | - |
| D-PSMa3 | + | + | - |
| L-PSMa3 | + | + |
30 min |
| D-PSMa3 | + | + | 25 min |
| - | -/+ one for each duplicate | - |
Incubated all samples for 1h 30m min (including the 30 min preincubation step with or without PSMa3) at 37°C.
Measured at A360ex/A460em, over a timecourse of 2h, 37°C, every 10 minutes for 2h.
27.09.2019
Thawed lysate from 26.09. from -80°C and performed new proteasome assay as before:
- water + fluorophore
- D-PSMa3 20uM + fluorophore
- water - fluorophore
Measured in the platereader as before for 1/2 every 5 min.
No fluorecence detectable.
28.09.2019
seeded HEK293 cells in 3 wells of a 6-well plate
29.09.2019
Washed and harvested HEK293 cells as before.
Lysis, centrifuged lysates at 16000g at 4°C for 10 min.
Loaded black plates as follows.
| protein (20uM) | LLL-AMC | ATP |
| - (water) | + | + |
| L-PSMa3 | + | + |
| D-PSMa3 | + | + |
| L-IZN17 | + | + |
| D-IZN17 | + | + |
| - (water) | + | - |
| - (water) | - | + |
Incubated lysates shortly on ice then added 25 uL lysate to each well.
Measured in the plate reader at 37°C as before.
09.10.2019
Performed proteasome assay as before with Jurkat T- cells and HEK293 cells. Used IZN as peptides and PMSF as control.
14.10.2019
Proteasome assay with water, L-IZN, D-IZN with or without PMSF. With PMSF: duplicates, without triplicates. HEK293 cells, measured as before.
