Anti-FLAG Test PO Extractman
Date: 10/02/2017
Cell line: HEK293_STAT3-3xFLAG (6/30/17)
Use recycled beads conjugated with anti-FLAG (SIGMA F1804)
Scale: 50mg per experiment, with 5ul of beads
Extraction/wash buffers:
Samples will be:
2 replicates for each condition will be processed on the Extractman, 2 replicates per condition will be processed by hand.
Weigh out 4 - 100mg aliquots of powder. Complete one set before moving onto the next set.
Take the powder out from liquid N2 and leave the tube @ RT for 1’
Add 400ul buffer with protease inhibitors to powder, mix by vortexing, try to let the powder resuspend completely – typically takes ~10 sec; no more than 30 sec. Do not let the tube come to room temperature.
Sonicate 25x 2 sec @ 2 Amp
Combine tubes after sonication before spin
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Wash beads 3x with appropriate buffer on magnetic rack as usual
Divide the lysate to set up IPs using 5ul of recycled anti-FLAG beads per 50mg (8 total conditions – 4 by hand, 4 by Extractman); each IP ~222ul
Incubate @ 4°C for 30’
2 sample sets per replicate by hand; 2 sample sets per replicate via Extractman
Extractman samples:
Place microplate on Extractman base Fill twelve wash wells (columns 3,4,5) with 100ul of appropriate buffer per well Fill 2 elution wells (column 6) (1 per buffer condition) with 15ul of 1.1x LDS Fill 2 elution wells (column 6) (1 per buffer condition) with 15ul of FLAG peptide (Apex Bio A6001) Make sure all wells have a convex meniscus of about 2mm.
Position release magnet under the first wash well column (column 3) Resuspend the beads and transfer 250ul of the IP reaction to the 230ul input well column (column 2) on the microplate (overfilling is intentional to create a prominent meniscus)
Secure bead strip capture strip to Extractman handle, then position handle onto base Move handle over column 2 to collect the beads. Hold briefly until all beads are recovered.
Move handle to first wash well column. The beads will fall to the bottom of the wash well. Hold briefly until all beads have dropped.
Repeat prior 2 steps for remaining wash wells
Move release magnet to elution well column; beads will rise from final wash column to bead capture strip; hold briefly until all beads are recovered
Move handle to elution well column, beads will fall from capture strip to elution well; hold briefly until all beads have dropped.
After 10’ incubation, move release magnet away from elution well; beads will rise from elution well back to bead capture strip
Gently slide handle off base and recover beads from bead capture strip; save for potential analysis
Recover elutes from elution wells for analysis
Samples by Hand
Wash 3x 1ml extraction buffer (transfer the beads to fresh tubes during 2nd wash)
Elute 1 of each sample condition with 15ul of 1.1x LDS @ RT for 10’ (no shaking) and 1 of each sample condition with 15ul of FLAG peptide (Apex Bio A6001) @RT for 10’ (no shaking)
Collect the elutions (“E”)
ALL SAMPLES
Second elution from beads @70C with shaking for 5’
Add 0.8ul of 500mM DTT to each sample (final concentration ~25mM)
Heat samples @ 75C for 10’
Collect the elutions (“70C”)
Load elution for Coomassie stain
Run 20-well 4-12% Bis-Tris gel, stain with Coomassie
Coomassie gel:
- Marker_5ul
- 150_Exctractman_LDS
- 150_Extractman_FLAG
- 150_Hand_LDS
- 150_Hand_FLAG
- 300_Extractman_LDS
- 300_Extractman_FLAG
- 300_Hand_LDS
- 300_Hand_FLAG
- Space
- 150_Exctractman_LDS_70C
- 150_Extractman_FLAG_70C
- 150_Hand_LDS_70C
- 150_Hand_FLAG_70C
- 300_Extractman_LDS_70C
- 300_Extractman_FLAG_70C
- 300_Hand_LDS_70C
- 300_Hand_FLAG_70C
- BSA_50ng
- BSA_150ng
