N2102EP L1 screen in 24-well format
Date: 03/02/2021 - 03/03/21
Powder: N2102EP RU August 7, 2020
Extraction buffers (with protease inhibitors), see
Scale: 50mg per sample Buffer: 450ul per sample Beads: 10ul of anti-ORF1 or mIgG control beads slurry (do anti-ORF1 and mIgG IP on two different days
6 conditions screen with replicates (4 replicates a-d) and controls; 4 experimentals + 4 controls: 8x 2.5ml = 20ml buffer for each; make extra (50ml of each):
(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
Prepare the following:
Solvent Plate 2.5ml 96-well deep-well microplate Add 2.2ml of each extraction buffer in corresponding well Keep the plate @ RT before extraction
PI Plate 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 10ul of anti-ORF1 beads or mIgG control to each well (10ul 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 18ul of 40mM Tris pH 8.0, 2% SDS
Use individual low-bind tubes to collect the eluate instead of loading plate
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 We did not check temperature this time
*No additional time was added to this row.
We check the relationship between sample temperature and total energy output using a mock plate with same samples setting (24 samples, 50mg each with 450ul of buffer), rows on either side of sample rows were filled with water. 1st and 2nd sample rows (total energy given ~150J): temperature increased from 3-4°C to 4-5°C and 3rd sample row (total energy given 211J): temperature increased from 4-5°C to 8-10°C. Lesson learned: we should never let total energy output exceed 200J at a time in order to minimize temperature change. If that happens and sonication time of a particular row is much shorter than others, chill plate on ice before continue.
—> 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
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
Lane 6-9: buffer 9
Lane 10-13: buffer 10
Lane 14-17: buffer 11
Lane 18-21: buffer 13
Lane 22-25: buffer 17
Lane 26: 50ng BSA+2.5ul Prestained marker
Run this gel when both screens are done
Anti-ORF1 gel: 1 second exposure
mIgG gel: 1 second exposure
Prepare MS samples using S-trap Ultra-High Recovery protocol
For details see wiki entry December 3, 2020 - N2102EP L1 screen in 24-well format last section “Sample preparation_ultra high recovery protocol” December 3, 2020 - N2102EP L1 screen in 24-well format
Difference from prep done in last December”
After elution, before drying down the samples, split each sample in two fractions: 75% for LFQ and 25% for TMT.
Samples are label either anti-ORF1 or mIgG and numbers 1-24 (“L” for LFQ and “T” for “TMT”)
1-4: buffer condition 1 (4 replicates a-d)
5-8: buffer condition 9 (4 replicates a-d)
9-12: buffer condition 10 (4 replicates a-d)
13-16: buffer condition 1 1(4 replicates a-d)
17-20: buffer condition 13 (4 replicates a-d)
21-44: buffer condition 17 (4 replicates a-d)
Samples were sent to NL on March 16, 2021