N2102Ep, NTERA and CRC anti-ORF1 and anti-ORF2 IPs with controls
https://docs.google.com/document/d/1w0r2hDzVwqZUBwKXDx_mFUOd6fqNh_VJQ-5FXvBBQUo/edit?usp=sharing
Date: 12/15/20 -12/19/20
100mg scale with 20ul beads
Cell lines and CRC tissue
1) N2102Ep
2) NTERA
3) 185T (CRC, high ORF1p expresser; see wiki entry “June 10, 2020 - CRC (162_185_197_197_268_284) anti-ORF1 IP”)
4) 284T (CRC, low ORF1p expresser)
5) MT302 (positive control)
6) HeLa Kyoto (negative control)
Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Prepare extraction buffer with protease inhibitors (1:100); Add 1:4 (w/v) of extraction buffer to powder. Add RNasin to extraction buffer (1:40 for tumor tissue and 1:250 for cells) and wash buffer (1:250 for tumor tissue and 1:1000 for cells)
Weigh out 4x 100mg of each powder
4x 100mg each; 2x 100mg for anti-ORF1 IP and 2x 100mg for anti-ORF2 IP
Will do anti-ORF1 and anti-ORF2 IP and two different days
Take 2x 100mg of each first and do anti-ORF1 IP; Keep 2x 100mg in -80C freezer and do anti-ORF2 IP the next day
Sonicate samples for 5x 2 sec at 2 Amp (energy output 100mg samples: ~20J)
Spin @ 20k rcf, 4°C for 10' (Eppendorf Centrifuge 5417R);
Take 20ul of clarified whole cell lysate (input) from each replicate; Add 5ul of 10% SDS (final concentration will be 2% SDS)
Snap freeze and dry the samples completely in the speed vac. Keep the pellets @ -80C and send to NL for MS
Take another 5ul of input from each replicate and pool the input from duplicates of the same tissue or cell line (10ul total). Use this for Bradford assay and load 25ug of input for Western
Set up 100mg anti-ORF1 or anti-ORF2 IPs (duplicates for each cell line/tissue)
IP @ 4°C for 30'
Wash beads 3x 1ml with extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute in 25ul of 2% SDS/40mM Tris, pH8 @ 70°C for 5' with mixing
Collect elution for Western and MS
• 12.5% for Western; combine two replicates; will be 25mg equivalent at the end,
• 87.5% for MS; two replicates; Snap freeze; Dry samples down in speed vac; Keep @ -80C and then send to NL
For CRC high ORF1p expresser, ORF1p signal from 25mg anti-ORF1 IP is similar to 5mg MT302 anti-ORF1 IP (see wiki entry see wiki entry "June 10, 2020 - CRC (162_185_197_197_268_284) anti-ORF1 IP")
For Western samples, add 50mM DTT and 1x LDS (all final concentrations)
Heat samples @ 70°C for 10'
Load all elution on one gel
Load 25ug of lysate (2.5ug of MT302)
Load 3ul of lysate (1/10 vol of MT302)
Bradford assay of cell lysate
For reference, see wiki entries
June 16, 2020 - Western Blots of Lars' samples
July 28, 2020 - Anti-ORF1 IP with N2102Ep and NTERA2
October 20, 2020 - N2102Ep and NTERA anti-ORF2 IP for Western
12/19/19 - PA-1, N2102Ep and NTERA2 anti-ORF1 IP
Gel 1 for all elutions
1) Marker
2) E_N2102Ep_anti-ORF1_25mg scale
3) E_NTERA_anti-ORF1_25mg
4) E_185T_anti-ORF1_25mg
5) E_284T_anti-ORF1_25mg
6) E_MT302_anti-ORF1_2.5mg; positive control
7) E_MT302_anti-ORF1_12.5mg; positive control
8) E_HeLa Kyoto_anti-ORF1_25mg negative control
9) Marker
10) E_N2102Ep_anti-ORF2_25mg scale
11) E_NTERA_anti-ORF2_25mg
12) E_185T_anti-ORF2_25mg
13) E_284T_anti-ORF2_25mg
14) E_MT302_anti-ORF2_2.5mg; positive control
15) E_HeLa Kyoto_anti-ORF2_25mg negative control
Gel 2 for cell lysate (same amount, 25ug)
1) Marker
2) Input_N2102Ep_anti-ORF1_25ug
3) Input_NTERA_anti-ORF1_25ug
4) Input_185T_anti-ORF1_25ug
5) Input_284T_anti-ORF1_25ug
6) Input_MT302_anti-ORF1_2.5ug
7) Input_HeLa Kyoto_anti-ORF1_25ug negative control
8) Marker
9) Input_N2102Ep_anti-ORF2_25ug
10) Input_NTERA_anti-ORF2_25ug
11) Input_185T_anti-ORF2_25ug
12) Input_284T_anti-ORF2_25ug
13) Input_MT302_anti-ORF2_2.5ug
14) Input_HeLa Kyoto_anti-ORF2_25ug negative control
15) Marker_1ul
Gel 3 for cell lysate (same volume, 3ul; MT302 1:10 diluted)
1) Marker
2) Input_N2102Ep_anti-ORF1
3) Input_NTERA_anti-ORF1
4) Input_185T_anti-ORF1
5) Input_284T_anti-ORF1
6) Input_MT302_anti-ORF1_3ul (1:10 diluted lysate)
7) Input_HeLa Kyoto_anti-ORF1
8) Marker
9) Input_N2102Ep_anti-ORF2
10) Input_NTERA_anti-ORF2
11) Input_185T_anti-ORF2
12) Input_284T_anti-ORF2
13) Input_MT302_anti-ORF2_3ul (1:10 diluted lysate)
14) Input_HeLa Kyoto_anti-ORF2
15) Marker_5ul
Wet transfer, 70V for 2h
Block the membranes in TBST/5% milk @ RT, 2hr
Cut the membrane in half, between 75 and 50KD
Elution blot:
Upper panel: Rabbit anti-ORF2 Western (Clone 5)
Primary Ab: Rabbit anti-ORF2 antibody (Clone 5, 1.03mg/ml), 1:500, 4°C, overnight
Secondary Ab: ECL anti-rabbit HRP 1:5,000, RT, 1hr
Lower panel: Anti-ORF1 Western (Abmart, 4H1, 2mg/ml)
Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
10 seconds exposure, high sensitivity, auto tone
3 minutes exposure, super sensitivity, auto tone (top panel only)
Lysate blots:
Lower panel: Anti-ORF1 Western (Abmart, 4H1, 2mg/ml)
Primary Ab: Mouse anti-ORF1 antibody, 1:1,000, 4°C, overnight;
Secondary Ab: ECL anti-mouse HRP 1:5,000, RT 1hr
30 seconds exposure, high sensitivity, auto tone
1st image: 25ug of lysate (except 2.5ug of MT302); 2nd image: 3ul of lysate (except 3ul of 1:10 diluted MT302 lysate)
5 minutes exposure, super sensitivity, auto tone
1st image: 25ug of lysate (except 2.5ug of MT302); 2nd image: 3ul of lysate (except 3ul of 1:10 diluted MT302 lysate)
