HEK 293 PPR6-3xFlag cell line was moved from adherent condition to suspention according to this paper From “Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells” Domanski, LaCava 2017. Cell were induced by different concentration of DOX. I harvested cell ~ 10 million cells per sample, 6 conditions - 2 replicate
| label | dox concentration | sample concentraion by BCA assay (ug/ul) | ||||
| 0 | 0 | 1,97 | ||||
| 1 | 1 | 1,61 | ||||
| 2 | 2 | 3,4 | ||||
| 3 | 3 | 2 | ||||
| 4 | 4 | 2,63 | ||||
| 5 | 5 | 3,8 | ||||
| 0* | 0 | 1,89 | ||||
| 1* | 1 | 1,78 | ||||
| 2* | 2 | 3,15 | ||||
| 3* | 3 | 2,3 | ||||
| 4* | 4 | 2,58 | ||||
| 5* | 5 | 4,22 |
Spin 1000 rcf 4C for 5’ Remove the sup, resuspend in 2 ml of DPBS, spin again
Resuspend in 500 ul of extraction buffer (RT) with protease inhibitor (5 ul of inhibitor for 500 ul of buffer). Totally for 6 samples you need 3000 ul +30 ul of pr.inh. Do 3400 ul + 34 ul to cover pipetting error. 12 samples - 6800 ul + 68 ul
Sonicate 5x2 sec @4 amp twice.
Spin max speed 4C for 10’ Transfer the supernatant to the fresh tube (“SUP”)
Resuspend each pellet in the same volume of 1.1 LDS as a volume of appropriate sup fraction. Incubate @70C for 5’
Spin 20k rcf, 25C for 5’ Keep the supernatant as “pellet”.
For these blots I used only * samples (SUP fraction). There is 35 ug per well of sample.
As a control I used parental cell line - 7,63 ug/ul
Hua's SUP 8,91 - ug/ul
Leila's powder (adherent HEK 293 with RRP6 3xFlag) - 7,12 ug/ul.
RRP6-3xFlag control - n.el from IP in 300 mM NaCl conditions (used 1,5 - it's 5% of elution, total elution was 30 ul)
Two gels run in the same tank, same time, voltage, blocked in 5% milk overnight.
Ab concentration 1:1000
Comments from Nastya
3x-Flag signal starts from 1 ng/ml and there no pattern when concentration of dox increases.
It’s impossible to tell where is a band of RRP6 without 3xFLAG on this blot
Suggestion - change Ab or use commercial pure RRP6 protein as a control