CRC tumor L1 screen_July 2021
6 conditions screen with 4 replicates:
(1) 500 mM NH4 Acetate pH 7.0, 250 mM NaCl, 1% v/v Triton x-100
(9) 20 mM HEPES 7.4, 300 mM NaCl, 1 mM Sarcosyl
(10) 20 mM HEPES pH 7.4, 50 mM MgCl2, 0.1% v/v Tween 20
(11) 20 mM HEPES pH 7.4, 100 mM MgCl2, 0.1% v/v Tween20
(13) 20 mM HEPES pH 7.4, 300 mM K acetate, 0.1% v/v Tween 20
(17) 20 mM Tris pH 8.0, 300 mM Na3 Citrate, 1% v/v Triton x-100
L1 screen conditions (same conditions were used for N2102Ep screen)
March 2, 2021 - N2102EP L1 screen in 24-well format (4x 6 conditions)
2 tumors were picked 162 and 197
Choose CRC tumor with matching controls based on previous anti-ORF1 Western
11/19/18 - 162T and 164N anti-ORF1 PO for Western (Elution only)
June 10, 2020 - CRC (162_185_197_197_268_284) anti-ORF1 IP
June 17, 2020- CRC (159_185_174) anti-ORF1 IP
2 types of IP will be done for each tumor
Experimental: CRC tumor anti-ORF1 IP
Negative controls: matching normal anti-ORF1 IP or CRC tumor mIgG
Protease and RNase inhibitors
RNasin® Ribonuclease Inhibitor (Promega N2515, 40u/ul, 10,000U, 250ul per vial)
Extraction buffer: Protease inhibitors (1x, see PI plate) plus RNasin 1:250 (450ul per sample; sample buffer in 4 wells; 6 buffer conditions; 3 types of IP per sample; 2 types of tumor tissue: 450x (4x6) x 6 / 250 = 259.2ul RNain will be needed for extraction buffers.
Prepare extraction buffer with RNasin right before the experiment starts
Since the RNasin concentration in extraction buffer and wash buffer will be different, prepare extraction buffer and wash buffer separately
For each screen: take 2ml of each extraction buffer (~10% over 4x450ul) in a 5ml tube, add 8ul of RNasin (1:250) and 20ul of 100x protease inhibitors; mix well; transfer 500ul of each extraction buffer to 4 adjacent wells of the Solvent Plate (4 replicates per buffer condition); After adding 450ul of extraction buffer to cell powder, there will be 50ul of extraction buffer left in each well of the Solvent Plate; use this 50ul of extraction buffer to equilibrate beads in the binding plate before IP
Prepare wash buffer
Prepare 5ml of each kind of wash buffer (~5% over 4x 1200ul): add 5ul of RNasin (1:1000) and 25ul of 100x PI (1:200; I used less 50% less PI in wash buffer); mix well; transfer 1250ul of each wash buffer to 4 adjacent wells of the Solvent Plate. This is done after extraction buffer was added to the powder and beads. 3 wash steps : 500ul x2 and 200ul->1200ul total.
Scale: 50mg per sample
Buffer: 450ul per sample
Beads: 5ul of anti-ORF1 (tumor and normal) or mIgG control (tumor only) beads slurry (do one 24-well screen per day) 5ul beads should be sufficient for 50mg CRC anti-ORF1 IP
May 5, 2021 - Effects of ZnCl2 on IP specificity using CRC tissue (see Western result of the anti-ORF1 beads titration test at the end of the wiki entry)
6 conditions screen with replicates (4 replicates a-d) and controls; 4 experimental (tumor) + 8 controls (mIgG and normal tissue): 12x 2.5ml = 30ml buffer for each; make enough for 2 tumors (make 100ml of each buffer):
Prepare the following:
Solvent Plate
2.5ml 96-well deep-well microplate
Add 500ul of each extraction buffer in corresponding well
Keep the plate @ RT before extraction
PI Plate (skip this, PI will be added to extraction buffer directly)
0.8ml 96-well deep-well microplate
Add 5ul of 100x protease inhibitor in each well (5ul x24)
Keep the plate @ 4°C
Label 1.5ml eppendorf tubes 1-24; Keep the tubes on ice
Cleared extract will be transfer to these tubes after sonication and centrifugation
Keep the tubes on ice
Binding Plate
0.8ml 96-well deep-well microplate
Add 5ul of anti-ORF1 beads or mIgG control to each well (5ul x24)
Equilibrate beads in each well with 100ul of corresponding buffer before IP
Keep the plate @ 4°C
Elution Plate
96-well PCR plate
After IP, wash the beads with 200ul of extraction buffer
At the last wash, transfer beads to elution plate
Get rid of the buffer and elution with 25ul of 40mM Tris pH 8.0, 2% SDS
Use individual low-bind tubes to collect the eluate instead of loading plate (Take 20% or 4ul of elution for gel and keep the rest in -80C. The remaining 80% of each sample will be used for S-trap)
Protocol:
Dispense 50mg of power to 24 wells to 0.8ml 96-well deep-well hold on metal plate merged in liquid N2
Move the 96-well plate with samples at RT ~1 min
Extraction @ 1:9 w:v (450ul of buffer per 50mg of powder);
Add H2O to the rows on each side of the sample rows
• water row
• sample row 1
• sample row 2
• sample row 3
• water row
In order to eliminate issues related to heating and cooling of the probe itself the following should be done:
• move the probe into cold room at least 1hr in advance
• run the probe in H2O in the cleaning trough once for 30 sec before starting the procedure
Sonication: use 1 Amp, applying 30 sec to each row
—> Try 30 sec should (record all J applied to each row)
—> using the temp prob - check the temp of the solutions using a middle well - record - give it 5 sec to equilibrate (may want to check and edge and a middle well to see that they yield results within 1-2 degrees)
—> after sonication, do this again on the same well - record
Do one screen per day. Try tumor with mIgG first.
General practice:
Try 30sec sonication @ 1 Amp first, make sure the average energy was below 250J per row. The temperature increase should not exceed 2-3C after sonication.
If additional sonication is needed, put the plate on ice to let it chill first and then add another 10sec (~50J).
For the first 30 sec sonication, if the total energy reached 250J and there is still time left, stop sonication, go to the next row or put the plate on ice before adding another 10 seconds.
—> clean the probe tips with a 5 sec zap in water between runs, the plate should be on ice while this happens - always on ice when not being sonicated - with a kimwipe dab away all excess liquid after probe cleaning
—> after applying 30 sec to each row - visually inspect - probably some wells will need more sonication. Apply an additional 10 sec to each row (treat them all the same) - check again. Once the samples look like a milky/semi-transparent homogenate, you are done - remove the rubber may carefully and use kimwipes to sop up any liquid the is between the wells (to avoid cross mixing etc) - transfer sample to cold microfuge tubes and centrifuge
—> total sonication time is expected to be ~40-50 sec - should not exceed 1 min (total energy should be ~250J or below)
Transfer the lysate to 1.5ml Eppendorf tubes
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
During the spin wash the beads in respective buffers
Transfer the cleared extract to the tubes containing the pre-washed beads
IP @ 4°C for 30’
Spin the plate down @ 3k rpm for 1’
Wash beads with 2x 500ul of corresponding extraction buffer by moving the binding plate around on the magnet
Add 200ul of extraction buffer to the beads
Transfer the beads to Elution Plate
Put the Elution Plate on magnet and get rid of the buffer in each well
Elution with 25ul of elution buffer (40mM Tris pH 8.0, 2% SDS)
Elute @ 70°C for 5’ with shaking
Collect eluates in protein low-bind tubes
Take 5ul (20%) of each eluate for Sypro gel (equivalent to 10mg); Run each replicate separately
Remaining samples dry down in speed vac and will be used for S-Trap MS prep
For gel samples, add 50mM DTT and 1x LDS (final concentrations)
Heat all samples @ 70°C for 10’
Load two 26-well 4-12% Bis-Tris gels (Sypro stain); one gel for anti-ORF1 IP and the other one for control mIgG
Lane 1: 1ul unstained marker
Lane 2-5: buffer 1 (sample # 1-4)
Lane 6-9: buffer 9 (sample # 5-8)
Lane 10-13: buffer 10 (sample # 9-12)
Lane 14-17: buffer 11 (sample # 13-16)
Lane 18-21: buffer 13 (sample # 17-20)
Lane 22-25: buffer 17 (sample # 21-24)
Lane 26: 50ng BSA+2.5ul Prestained marker
162T anti-ORF1 (1s exposure)
162N anti-ORF1 (1s exposure)
162T mIgG (1s exposure)
197T anti-ORF1 (1s exposure)
197N anti-ORF1 (1s exposure)
197T mIgG (1s exposure)
Repeat 50mg IP manually
50mg powder, 450ul extraction buffer, 5ul beads;
Manual screen (1s exposure)
Loading:
Unstained marker_1ul
1: 162N anti-ORF1_buffer 1
2: 162N anti-ORF1_buffer 17
3 & 4: 197T mIgG_buffer 10
5 & 6: 197T mIgG_buffer 17
BSA_25ng
BSA_50ng
Prestained marker_2.5ul
Prepare MS samples using
Wiki protocol MS sample preparation using S-Trap Micro Ultra-High Recovery protocol rev. 05/27/21
Skip steps 1 and 2 in the above protocol, do the following instead:
Take the dried elution (80%; 20ul of 2% SDS/40mM Tris, pH8) from -80C freeze, resuspend each pellet with 23ul of 3.5% SDS, 8 M urea, 100 mM glycine* pH 7.55 (final SDS concentration will be little over 5%)
Follow steps 3 to 14 of the Wiki protocol
Before step 15, dry down in speedvac, split each sample in two tubes: 75% for LFQ and 25% for TMT
Two tubes of the same sample will be labeled the same way except tube containing fraction for TMT will have a red “T”
Dry samples to Luciano will be sent the NL
Here's the wiki entry of the MS runs from the last screen made by Hua (Tumors 162 and 197): https://macromolecule-child.rockefeller.edu/#August%202021%20%E2%80%93%20Hua_ORF1%20screen%20in%20tumors