N2102EP Quadruplicate Anti-ORF1 IP
Date: 28 May 2021
Prior to beginning, place sonicator arm in cold room to allow it to equilibrate to 4C. Get a cooler with LN2 and precool tubes for powder, weigh tools and large tweezers. Get a bucket of ice to cool your wash buffers. Samples are to be kept on ice at all times unless otherwise noted.
Experiments will be performed by Apotolis (8x2), Leila (8x4), and Shaoshuai (8x2).
Each person will work with one buffer at a time to reduce capacity for error in wash steps.
Cell Lines:
- N2102EP no treatment
- N2102EP DMSO control
- N2102EP Drug 12 treatment
- N2102EP Drug 473 treatment
Extraction/Wash buffers:
- 20mM HEPES Na pH7.4, 500mM NaCl, 1% Triton X-100 (buffer L1)
- 20mM HEPES Na pH7.4, 50mM MgCl, 0.1% Tween 20 (buffer 10)
- 20mM HEPES K pH 7.4, 300mM KAcet, 0.1% Tween 20 (buffer 13)
- 20mM TrisCl pH 8.0, 300mM Na3Cit, 1% Triton X100 (buffer 17)
Wash buffers should be kept on ice; extraction buffer with protease inhibitors/RNAsin kept at RT.
Prep extraction buffer with RNAsin (1:250) and protease inhibitors (1:100); Add 1:4 (w/v) of extraction buffer to powder. NOTE: These are ONLY added to extraction buffer, NOT WASH BUFFER
Add 28ul of RNAsin and 70ul of Protease inhibitors to 7ml extraction buffer (RT) 96ml extraction buffer/wash buffer for wash (in ice)
Label and precool 64x2ml safe lock eppendorf tubes in LN2
Weigh out 16x100mg of powder for each cell type (64 samples total)
Allow powder to sit at RT for 1 minute before adding buffer
Add 400ul of extraction buffer (RT, with protease inhibitor/RNAsin added) to each sample, Vortex to resuspend. If powder is not resuspended after 10 seconds, return sample to ice for 30 seconds before additional vortexing to prevent temperature increase
Sonicate 5 x 2 Seconds, 4 Amp, expected energy output is 15J - 20J; Record output readings for each sample
Spin @ ~21k rcf (top speed of benchtop eppendorf 5424R), at 4C for 10 minutes. ,
Wash beads while spinning
Place empty 1.5 ml safe-lock tubes on magnet; add 1ml of appropriate wash buffer to tubes
Add 20ul anti-ORF1 beads to each tube
Close tubes and remove tubes from magnet, vortex and quick spin to get beads from top of tube. Aspirate buffer with vacuum 2x 1ml additional washes (3 total)
After spin, save 20ul of clarified whole cell lysate (Input).
Use remainder of clarified whole cell extract to setup 64x IPs
IP incubation with end-over-end mixing in cold room for 30 min
Wash the beads with 3x1 mL of appropriate wash buffer.
Switch beads to new tubes during second wash step to ensure nothing carries over from IP to eluate.
After the 3rd wash, give beads a final quick spin to remove last ul of wash buffer
Elute in 25ul of 2% SDS/40mM Tris, pH8, with mixing, 1000rpm, 70C.
Collect the elution on Magnet.(Elution)
Save 2.5ul aside (10% of 100mg IP) of each elution for gel analysis.
Combine the 10% elution of 4 replicates from the same cell line (~ 40mg IP) for blue-silver gel analysis. Hand remaining samples off to Dennis for S-trap preparation
GEL IMAGES (2.5ul of elution, Silver Staining)
