N2102EP L1 screen in 24-well format
Date: 12/03/20 -12/04/20
Powder: N2102EP RU August 7, 2020
Extraction buffers (with protease inhibitors)
Scale: 50mg per sample
Buffer: 450ul per sample
Beads: 10ul of anti-ORF1 beads slurry
24 conditions in duplicate = 48 samples total; Perform 24 samples at a time (12 buffer conditions in duplicates: 1a, 1b to 12a, 12b)
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 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 18ul of 1.1x LDS
Use individual low-bind tubes to collect the eluate
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); # 22-24 has 2mM Glutaraldehyde in the extraction buffer, but not in the wash buffer
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
—> 30 sec should yield between ~250 - 280 J (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
—> 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
Conditions with 500mM NaPhosphate (#19-23), when wash with extraction buffer (all without GA), NaPhosphate precipitated out, crystal started to form in the wells; wash two more times with 1x PBS to get rid of the crystal before transfering the beads to elution plate
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 2.5ul (10%) of each eluate for Sypro gel (equivalent to 5mg); Combine the duplicates (equivalent to 10mg per condition, no replicate), load 5ul on gel
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 26-well 4-12% Bis-Tris gel (Sypro stain) Lane 1: 1ul unstained marker Lane 2-25: buffer conditions 1-24 Lane 26: 50ng BSA+2.5ul Prestained marker
Run this gel when all 24 conditions are done.
Sample preparation_ultra high recovery protocol
Date: 12/10/20-12/11/20
24 samples were prepared as 2 sets of 12 samples per day. For steps 10-12, handling 6 tubes at a time.
1) Add 23 uL of 3% SDS, 8 M urea, 100 mM glycine pH 7.55 to the (dried down) samples.
50mg IP was eluted in 25ul of 2% SDS/40mM Tris; 2.5ul (10%) was taken for gel analysis. Remaining 22.5ul was dried and saved in -80°C for MS. When the pellet was resuspended in 23ul of 3% SDS, 8 M urea, 100 mM glycine pH 7.55; final SDS concentration will be 5%.
2) Reduce by adding 1 µL reductant (120 mM aq. TCEP) to sample (5mM final concentration) and mix.
3) Incubate 37°C for 15 minutes (Thermomixer at 600 RPM).
4) Put the tube on ice briefly to bring the temperature down to RT. Set Thermomixer to 47°C and humidify.
5) Alkylate by adding 1 uL alkylator (500 mM MMTS). (final concentration: 20 mM)
6) Incubate @ RT for 15 minutes.
7) Acidify by adding 2.5 µL 55% aq. phosphoric acid to the 25 µL sample. This is different than the normal protocol.
8) Place S-trap column into a standard 2.0 mL vial (cut-off lid).
9) Add 165 µL of S-Trap binding buffer (90% MeOH, 100 mM final TEAB, pH 7.55) into the S-Trap micro column.
10) The next two steps must be done as quickly as possible. Add 2 µg of trypsin/lys-C mix into the acidified sample, immediately mix by pipetting up and down, then immediately transfer the mixture into the S-Trap binding buffer held in the micro spin column. Again mix by pipetting up and down.
11) Spin in bench-top centrifuge, the S-Trap column in a standard 2.0 mL tube at 4000 g for 30 seconds
12) Wash by adding 150 µL S-Trap buffer (pH=7.55) to the spin column and centrifuging through (4000 g, 30 seconds). Remove flow through. Repeat three times. Change the direction of the tubes between washes
13) Place S-Trap column into a new 1.7mL Lo-bind tube.
14) Add 0.5 µg of trypsin/lys-C in 25 µL of 50 mM TEAB, pH 8 to the top of the protein trap 10x stock (5ug/25ul): Resuspend 20ug of trypsin/lys-C in 100 µL of 1 mM HCl; dilute to 1x working solution with 55 mM TEAB, pH 8.5 before
15) Cap the S-Trap column loosely and incubate for 2 hrs at 47 °C (with Thermomixer at 0 RPM). DO NOT SHAKE. The cap MUST NOT form an air-tight seal.
16) Add 40 uL of 50 mM TEAB pH 8.5 to the S-Trap column. Centrifuge elution through at 4,000 g for 30 seconds
17) Add 40 uL of 0.2% formic acid. Centrifuge elution through at 4,000 g for 30 seconds
18) Elute hydrophobic peptides with 35 μL 50% acetonitrile, 0.2% formic acid (v:v). Centrifuge elution through at 4,000 g for 30 seconds
19) Dry down in Speedvac (~120 minutes at RT). Store -80°C until ready for LC/MS.