Anti-ORF1 IP with N2102Ep and NTERA2
Date: 07/08/20
Cell lines:
1) N2102Ep_120419
2) NTERA_121019
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors and RNasin) 1:250 RNasin in extraction buffer and 1:1000 RNasin in wash buffer, no protease inhibitors in wash buffer
Scale and powder to beads ratio 100mg per IP reaction with 20ul of anti-ORF1(4H1) or LaORF1 beads; LaORF1 beads should undergo DMP XL before IP
XL nanobody to Epoxy beads after conjugation
Take aliquots of beads: 4x 20ul of anti-LaORF1-5 (08/01/19 and 10/24/19 mixed) wash once in 1ml of 1xPBS
Resuspend beads in 20mM DMP in Borate buffer (2ml per reaction with 100ul of beads; make this right before use when beads are washing; I am using more DMP solution than needed to ensure that the beads will be mixing well during the XL step below)
Perform XL reaction @ RT on the wheel for 30’.
After 30’, remove the XL solution
Wash beads with 1ml of PBST
Wash beads again with 1ml of TBST for 5’ @ RT
Wash beads again with 1ml of PBST
Wash beads with 1ml of 1x PBS
Wash all beads in 3x 1ml extraction buffer before IP
Anti-ORF1 IP
Weigh out 4x 100mg of each cell powder, add 400ul of extraction buffer to each tube (1:4 w/v), vortex to mix Among 4 replicates of the same powder, 2 will be used for anti-ORF1 IP and 2 for LaORF1 IP
New sonicator: Sonicate 5 x 2 sec at 2 Amp;
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 10ul for protein assay and Western (“Input”) Save 35ul of Input and add 35ul of Trizol, snap freeze in liquid N2 for RNA extraction
Set up two sets of four 100mg IP reactions with 20ul anti-ORF1 or LaORF1 beads
4 x 100mg N2102Ep → 2x monoclonal → 2x nanobody
4 x 100mg NTER2 → 2x monoclonal → 2x nanobody
IP @ 4°C for 30’
After IP, Save the flow-through for Western (“FT”)
Wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
During 3rd wash, split the beads in each tube
1st set for Sypro gel
Transfer 50% of beads to another set of tube; elute in 20ul of LDS (E_before precipitation) The remaining 50% of the beads was eluted directly in 250ul of TRIzol
1a. N2102Ep_anti-ORF1_Sypro (50:50 split)
2a. N2102Ep_LaORF1_Sypro (50:50 split)
3a. NTERA_anti-ORF1_Sypro (50:50 split)
4a. NTERA_LaORF1_Sypro (50:50 split)
We need to compare the elution before and after precipitation to show the recovery. So, I am doing 50:50 split.
2nd set for MS → whole reaction eluted in 250ul of TRIzol
1b. N2102Ep_anti-ORF1_MS
2b. N2102Ep_LaORF1_MS
3b. NTERA_anti-ORF1_MS
4b. NTERA_LaORF1_MS
Vortex for 1min, place the tubes aside (can be on ice or RT, Trizol extract is stable at RT)
Separate aqueous and organic phase by phasemaker
If you need to pause - freeze samples in TRIzol, then resume later
Spin the Phasemaker tube at 16k RCF, 30 sec
Add 25ul water and then 50ul Chloroform to the Phasemaker. Do this immediately before adding TriZol elutions or (perhaps safer still) immediately after transfering elutions to the Phasemaker tube. Adding Chloroform/water too far in advance may cause a failure. Not adding the water will cause the PhaseMaker to fail and 'swallow' the aqueous portion of the TriZol reagent mix.
Collect the 250ul of Trizol elution from above
Mix by hand, vigorously for 15 secIncubate 2 min @ RT with end-over-end mixing
Spin 16k RCF, 4C (not important, Trizol extract is stable at RT), 5’ Use a gel loading tip, pass it through phasemaker to reach the bottom layer (organic phase); check the volume ~165ul
IF doing RNA extraction proceed directly - save the organic for later processing.
→ 1ul to separate tube for QC and rest (~5ul) for RNA-seq.
→ no INPUT total RNA was specifically was saved for this but that would be added back normally.
Precipitate protein from organic phase using AmAc/MeOH (8 samples total)
1st set for Sypro → 50% of beads eluted in 250ul of TRIzol
1a. N2102Ep_anti-ORF1_Sypro (50%)
2a. N2102Ep_LaORF1_Sypro (50%)
3a. NTERA_anti-ORF1_Sypro (50%)
4a. NTERA_LaORF1_Sypro (50%)
2nd set for MS → whole reaction eluted in 250ul of TRIzol
1b. N2102Ep_anti-ORF1_MS
2b. N2102Ep_LaORF1_MS
3b. NTERA_anti-ORF1_MS 4b. NTERA_LaORF1_MS
Add 200ul of cold 0.1M AmAc in MeOH (between 1 and 1.5 volume of the organic phase)
Mix by hand, vigorously for 15 sec
Spin 16k RCF, 4C, 5 min (The organic phase moved to the top.)
Transfer the top layer to a fresh tube
Add 625ul of 0.1M AmAc/MeOH; Final organic phase to 0.1M AmAc/MeOH ratio is 1:5
Mix by inverting, and the sample is incubated overnight at –20°C.
Precipitate was pelleted, 20k x g for 30' @ 4C
Mark the side of Eppendorf tube that the pellet should be at; carefully remove the supernatant
After centrifugation, wash pellet with1ml of cold 0.1M AmAc/MeOH; 20k rcf for 15' @ 4C
Wash pellet again with 1ml of cold 100% Acetone; 20k rcf for 15' @ 4C
Discard supernatant, the pellet is dried under vacuum.
Resuspend pellet with 20ul of 1.1x LDS or 25ul of SDS/Urea buffer for S-trap N2102Ep_anti-ORF1_LDS
→ 2ul of the 20ul LDS (10%) will go to western blot → Make SDS/urea/glycine “fresh” (glycine should be added just before use) → If you have to pause, freeze dried down samples BEFORE SDS/urea addition
1) N2102Ep_anti-ORF1_LDS
2) N2102Ep_anti-ORF1_SDS/Urea
3) N2102Ep_LaORF1_LDS
4) N2102Ep_LaORF1_ SDS/Urea
5) NTERA_anti-ORF1_LDS
6) NTERA_anti-ORF1_SDS/Urea
7) NTERA_LaORF1_LDS
8) NTERA_LaORF1_SDS/Urea
For samples in LDS (save 10% in -80C for western in case ORF1p cannot be detected by Sypro stain); Also save 10% of the LDS elution before MeOH precipitation
Add DTT to 50mM to LDS samples and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze the elution before and after precipitation (1a-4a), Sypro stain
1) Unstained Marker_1ul
2) N2102Ep_anti-ORF1_before
3) N2102Ep_anti-ORF1_after
4) N2102Ep_LaORF1_before
5) N2102Ep_LaORF1_after
6) NTERA_anti-ORF1_before
7) NTERA_anti-ORF1_after
8) NTERA_LaORF1_before
9) NTERA_LaORF1_after
10) BSA_25ng
11) BSA_100ng
12) Space
13) Prestained Marker_5ul
For pellets resuspended in 25ul of SDS/Urea buffer, prepare MS samples using S-Trap high recovery protocol. This will be 90% of IP elution after MeOH ppt same as the real samples from Lars’ previous experiment. We can actually give this set to Kelly to see what we can get. (Note: since the yield of MeOH precipitation is too low, we will not do MS this time. Run the 2nd set: 1b-4b (90% of elution) on Sypro gel Run 4-12% Bis-Tris gel to analyze the elution after precipitation (1b-4b, 90% of elution loaded), Sypro stain
1) Unstained Marker_1ul
2) N2102Ep_anti-ORF1_1b
3) N2102Ep_LaORF1_2b
4) NTERA_anti-ORF1_3b
5) NTERA_LaORF1_4b
6) BSA_25ng
7) BAS_100ng
Extract RNA from Input and Elution (not done, sample saved in -80C)