LaCava Research Wiki

For experiments performed in preparation for this, please see:

• 26 May 21_N2102EP Drug Assay Prep_Dynabeads Testing
• 17 May 21_N2102EP Drug Assay Cell Growth, Drug Dosage and Harvest
• 23 April 21_N2102EP Drug Assay Prep_Pilot Test of Media Volume Increase

For MS experiments performed with the resulting eluates from this IP, please see:

• 29/05/2021_S-Trap Micro Ulra High Recovery Protocol rev 5/27/21 (Rome IP buffer screening, part 1)
• 29/05/2021_LC-MS sample queue: Rome IP buffer screening, part 1
• 30/05/2021_S-Trap Micro Ulra High Recovery Protocol rev 5/27/21 (Rome IP buffer screening, part 2)
• 30/05/2021_LC-MS sample queue: Rome IP buffer screening, part 2

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:

1. N2102EP no treatment
2. N2102EP DMSO control
3. N2102EP Drug 12 treatment
4. N2102EP Drug 473 treatment

Extraction/Wash buffers:

• 20mM HEPES Na pH7.4, 500mM NaCl, 1% Triton X-100
• 20mM HEPES Na pH7.4, 50mM MgCl, 0.1% Tween 20
• 20mM HEPES K pH 7.4, 300mM KAcet, 0.1% Tween 20
• 20mM TrisCl pH 8.0, 300mM Na3Cit, 1% Triton X100

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 higher concentrations are ONLY added to extraction buffer, NOT WASH BUFFER

Prep wash buffer with RNasin (1:1000) and hold at 4C or on 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. Repeat wash step twice (3x 1ml total washes).

After spin, save 20ul of each clarified whole cell lysate (Input).

Use remainder of clarified whole cell extract to setup 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