Comments from Nastya
buffer preparation step:
19th buffer was really white and it was hard to filter it.
extraction step:
18th, 19th, 20th - the lysate started to separate. 18th, 19th, 21st, 22nd and 23rd buffers left a big pellet after centrifugation.
washing step:
18th buffer made a crystals while washing beads, so beads were washed with DPBS. 19th buffer was frozen touched with a tip, so beads for lysate with 19th buffer were also washed just with DPBS. 20th made crystals while washing beads - then beads were washed with DPBS.
glutaraldehyde:
22nd, 23rd and 24th buffers were with glutaraldehyde only for extraction and the protease inhibitor wasn't used here. All washing steps for these conditions were made without glutaraldehyde.
glutaraldehyde tips: use cold water to disolve it.
This report was added to WIKI by Dennis Nanninga due to Nastya's leave.
N2102EP 24-well Hand Screen
Powder: N2102EP ERIBA April 2020
Extraction buffers (with protease inhibitors), see Excel sheet here
Scale: 50mg per sample
Buffer: 450ul per sample
Beads: 10ul of anti-ORF1 beads slurry
(comment: when done by hand, use 1 : 4 (w/v)(add 200 uL buffer), when doing screen use 1 : 9 dilution(w/v))
24 conditions in duplicate = 48 samples total; perform 12 samples at a time
Protocol:
Dispense 50 mg of power to 12 pre-labeled locking 1.5ml tubes
Leave samples at RT ~1 min prior to adding extraction buffer
Extraction @ 1:9 w:v (450ul of buffer per 50mg of powder)
Sonication: Setting 4 Amp, 5x2 seconds ~ 15J (standard ERIBA setting)(comment: adjust when using smaller volumes)
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’
Wash beads with 3x 500ul of corresponding extraction buffer; change to new tubes on 2nd wash
After 3rd wash give beads a quick spin to remove any remaining wash buffer
Add 30ul elution buffer (40mM Tris pH 8.0, 2.5% SDS)
Elute @ 70°C for 5’ with shaking
Collect eluates in protein lo-bind tubes
Take 10% of each eluate for Sypro gel; combine the duplicates for total of 24 samples for gel
Remaining samples for S-Trap MS prep (comment: dry down 25 uL)
Add 50mM DTT to samples for gel
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: 100ng BSA+2.5ul Prestained marker
