Checking L1 expression of pMT646, 647 and pLD564
Date: 09/23/21
Constructs to test:
pMT646: ORFeus ORF2p-3xFlag (clean tags) CMV promoter in pCEP4 Puro (S221, low and high)
pMT647: ORF2p only ORFeus ORF2p-3xFlag (clean tags) CMV promoter in pCEP4 Puro
pLD564: L1RP ORF2p-3xFlag
Take 1 tube of 1ml spun-down pellet from each construct Add 100ul of extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl + protease inhibitors; vortex to resuspend
Sonicate @ setting 3 for 3 sec
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect clarified lysates
Measure total protein concentration by Bradford assay; load 25ul of total protein on gel, including two controls from June shipment pMT646 and pMT647, also include pLD401 light (used for EM study),
Add 1x LDS and 50mM DTT (final concentration) to all samples
Heat samples @ 70C for 10’ before loading (see table below for loading order)
Run 2 identical gels,15-well 4-12% Bis-Tris gel
1) Marker_5ul
2) pMT646 S221
3) pMT646 low
4) pMT646 high
5) pMT647
6) pLD564
7) pMT646 Old
8) pMT647 Old
9) LD401 Light
10) Marker_2ul
11) Marker_2ul (gel 2 only)
Western analysis
Wet transfer 70V for 1.5h
Block in TBST/5% milk @ RT for 2hr
Membrane 1: Cut the membrane between 75KD and 50KD markers
Anti-FLAG Western (top)
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 (bottom)
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
1 minute exposure, high sensitivity, auto tone
10 minutes exposure, high sensitivity, no adjustment
Membrane 2: Cut the membrane into 3 pieces, below 100KD and above 50KD marker
Anti-FLAG Western (top)
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-Lamin B1 Western (middle)
Primary Ab: rabbit anti-Lamin B1 (Cell Signaling #13435), 1:1,000, 4°C overnight
Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT for 1hr
Anti-PCNA Western (bottom)
Primary Ab: mouse anti-PCNA antibody (Santa Cruz, sc-56) 1:1,000 (old Ab that has been sitting in the fridge for years)
Secondary Ab: ECL anti-mouse HRP 1:10,000 1 minute exposure, high sensitivity, auto tone
10 minutes exposure, high sensitivity, no adjustment
All tif files of western can be found in this folder https://drive.google.com/drive/folders/19rZ14QJirtQVAXTgjGxZDxoBPutTUUAz?usp=sharing