LaCava Research Wiki

Mat adjustment to fit plate.

* Thermo Flat Matrix CapMats (catalog: 4412-11) is the best one which can balance the hole size and spraying. *drill a hole every mat well using needle, it can balance the air force making mat attaches plate without crevice, so the tips can insert into buffer as much as possible.

sonicate without powders

• 8-tip probe: 602-A
• volume: 500ul of extraction buffer
• add 500ul of cold water between each sample row.

IP-Screening

Materials: 37.5mg of STAT3-3xFLAG/each well

"Buffer" 500ul of L1 buffer (20mM HEPES, 500mM NaCl, 1% triton)

Bead 5ul of not good ant-FLAG bead

Procedure:

1. • SOLVENT PLATE: 2.2 ml of L1 buffer in the wells of a 2.5 ml 96-well deep-well microplate
   • LOADING PLATE: 1 μl of 1M DTT in a 8-strip PCR tube
   • BINDING PLATE: Prepare a 0.8 ml 96-well deep-well microplate with 100 μl of each extraction solution, Dispense 5 μl of magnetic affinity medium slurry in each of well
   • PI PLATE: Prepare protease inhibitors by dispensing 5 μl of 100x stock into each of 24 wells of a 0.8 ml 96-well deep-well microplate
2. Pre-cool the manifold setup, Cell powder should also be cooled on LN2. Dump cell powder onto the manifold surface and pack it into all the wells of the manifold using the packing tool. Excess powder is recovered using a spatula. A pre-cooled 0.8 ml 96-well deep-well microplate is placed on top of the manifold such that the openings of the wells of each device are aligned and face each other; and then the sandwich is inverted, spacers are removed, and it is given a firm tap from above (the underside of the manifold) – transferring the powders out of the wells of the manifold and into the wells of the 96-well plate (EXTRACTION PLATE). In case of any slight error or spillage during dispensing, a lint-free laboratory tissue paper or fine paintbrush can be used to brush away any powder that has accumulated between the wells of the deep-well microplate.
3. Remove the EXTRACTION PLATE from LN2 and allow it to stand ~2 minutes at RT. The powders will remain thoroughly frozen during this time. Add 500 μl (37.5mg cell powders) of each RT solvent from the SOLVENT PLATE to the PI PLATE – producing solvents including 1x protease inhibitors. Transfer this entire mixture to the concordant wells of the EXTRACTION PLATE
4. Cover the plate with a cap mat
5. Using an 8-channel pipette with adjustable channel width, transfer crude extracts from the EXTRACTION PLATE to 24 x 1.5 ml tubes and centrifuge for 10 min, ~20,000 RCF at 4 °C in a bench top microcentrifuge.
6. Transfer the supernatants (clarified extracts) to the BINDING PLATE and cap the plate with a cap mat or other liquid-tight plate seal. Resuspend the beads fully within each well by manually inverting the plate several times. Place the plate on a rotating wheel at moderate speed (just enough to prevent the beads from settling) for 30 min
7. Remove the BINDING PLATE from the rotating wheel and briefly spin down to collect all liquid within the bottom of the well: e.g., 1-2 min at 2k RPM and 4°C in a Beckman Coulter Allegra X-14R with SX4750 rotor and microplate carrier.
8. Place the BINDING PLATE on a 96-well deep-well magnet to retain the beads at the well sides. Then remove the depleted extracts and remove the plate from the magnet. After removing the solution, remove the plate from the magnet.
9. Wash 500ul*2
10. Add 200 μl of concordant extraction solvent to each well of the BINDING PLATE (wash #3). Pipette the solution in each well up and down several times to fully resuspend the beads and transfer them to a 250 μl 96-well PCR plate (ELUTION PLATE) placed on a 8-strip pcr tube. Removing the wash solution once the beads have adhered to the well sides.
11. Briefly spin down (as in Step 10) and place the ELUTION PLATE on the 96-well PCR plate magnet to remove any remaining wash solution from the prior step
12. Add 19 μl of 1.1x LDS loading buffer or other elution solution, 70C at 1400rpm for 5min.Re-equilibrate the plate to RT if heated. Briefly spin the plate to recapture all beads and loading buffer at the bottom
13. Place the ELUTION PLATE on the 96-well PCR plate magnet and transfer the elutions to a plate containing 1 μl 1 mM DTT (LOADING PLATE).
14. Load all samples on a 26-well 4-12% Bis-Tris midi-gel and stain with silver blue Coomassie

Lane1: 5ul ladder

Lane2-25: 24 replicates (for lane2,3, lost some sample when loaded to gel)

Lane26: 100ng BSA