the assay lined out is to analyse the expression timecourse of
any recombimant protein in an adherent culture, it describes the assay
done in a six well plate, but you can use any format you like. wether you
use S9 or High five cells depends on your personal preferences, to make
a complete optimization you might wonna analyze both of them.
As a starting point I'll describe the use of a MOI of 5 and 10, check also other MOIs to reach for an optimal expression protocoll, remember that you can't extrapolate from one proteinl to another.
1. Seed two six well plates at a density of 106 cells per well. The final volume of media should be about 1-1,5 ml, this keeps the virus concentrated and faciliates the handing of the supernatant later.
2. Label plate#1 with MOI 5 and plate #2 with MOI 10 respectivly.
3. On each plate label wells #1-5 with the timepoints you want to analyze. normally as a start this will be; 24 h, 48 h, 72h, 96 h and 120h. This are the harvesting timepoints, you have only five timepoints, because the remaining wells serve as a uninfected control, label it as one either.
4. Take one plate and infect the cells at MOI 5 and take the other one and infect it at MOI 10. this means into the MOI 5 you put 5.000.000 pfu of your virus and into the wells with MOI 10 its 10.000.000.
5. Cover your plates and incubate at 27oC.
6. Harvest each well at the designated timepoint. Take supernatant and cells in a different tube. for the cells I recommend simply to wash them of with 1ml of icecold PBS or to wash them away together with the remaining media, thats what I do..
7. Pellet the cells at 800xg for ten minutes at 4oC, keep on ice.
8. Take of supernatant and transfer it to a seperate tube.
9. Store the fractions at
-70oC until use.
Processing your Timepoint Samples
1. Thaw all fractions on ice.
2. Make up the lysis buffer you
want to use (e.g. PBS, 0.1% Tx100) Use 100ml
per 106 cells, add the protease inhibitors you will use also
in the real extraction .
Keep all the stuff on ice all time. Add the lysis buffer to each cell pellet, vortex each cel sample to break up thecells and begin lysis.
3 Incubate the cells 30-45 ' on ice, vortex every now and then to support lysis.
4. Centrifuge at 1000xg for
10' to pellet cell debris. Transfer the supernatant to a new tube. Analyze
the lysate and supernatant by western, use 10 ml