Checking L1 expression of 9 L1 constructs
Date: 06/24/21
Constructs to test:
These are new plasmids made by Nastya (pMT646/647) and Samira (pMT868-870) as well as a number of our workhorse constructs from me and Lixin.
The goal of the experiment is to test the various new plasmids - they should perform as well or better than the old - and also make sure we can use our lab standard "Viral T" line with the new CMV promoter constructs, rather than using the 293T-LD line.
There are 24 tubes in RIPA buffer containing protease inhibitors which are lysates from the 6-well wells. Had I been a little smarter, I would have had a GFP+ control to show the transfection worked... but interestingly you can actually see it worked pretty well in that the L1 expression did seem to retard growth of both lines - but the tet-on constructs had less effect on protein levels in the viral T (which don't have the RTTA).
We ran a BCA assay and have concentrations for all of them. I suggest a normalized load (something like 5-10 ug per lane) for western.
Western loading (see end of the wiki entry, including samples from MT646 and 647 anti-ORF1 IP, 25ug cell lysate was loaded)
Note: protein concentration of 293TLD lysates was low. In order to load 25ug, take the desire volume from each sample, add 1x PBS to each sample, so the total volume will be 40ul for each. Put the diluted sample in speedvac and reduce the volume to ~10ul before add LDS and DTT.
https://drive.google.com/file/d/1ASMayFFmM4IurUeqETkuqpenI7BEkwbs/view?usp=sharing
Add 1x LDS and 50mM DTT (final concentration) to all samples
Heat samples @ 70°C for 10’ before loading (see table below for loading order)
Western analysis
Wet transfer 70V for 1.5h
Cut the membrane between 75KD and 50KD markers
Block in TBST/5% milk @ RT for 1hr
Anti-FLAG Western (upper panel)
Primary Ab: mouse anti-FLAG (Sigma F3165), 1:2,000, 4°C overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT for 1hr
Anti-ORF1 Western (lower panel)
Primary Ab: mouse anti-ORF1 from Abmart (4H1), 1:5,000, 4°C overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT for 1hr
10 second exposure, high sensitivity, auto tone
Image with label
Marty's comments:
1. All the new constructs appear to work
2. There is no meaningful difference between the Viral T and the 293T-LD cells with the CMV promoter constructs
3. The new CMV promoter constructs behave as well as the old tet-on ones do.
4. Of course the Viral T can't express the tet-on constructs (there's some crap in the pLD561 lane, but I think it's not ORF2)
5. The constructs work for IP and about half is 3xFlag elutable in these conditions - more for 647 than 646