Cell line pLD401 Completed in “RNA style” using nuclease-free reagents Scale: 800mg (4 x 200mg) Extraction buffer: 20mM HEPES, pH7.4, 1% Triton X-100, 500mM NaCl + P.I., + RNase Inhibitor @ 1:250 Wash buffer: 20mM HEPES, pH7.4, 1% Triton X-100, 500mM NaCl + P.I., + RNase Inhibitor @ 1:1000 Native elution at 500mM NaCl - detergent concentration may need to be lowered from 0.5% • 100mg of powder yields an estimated ~500ng of total protein via native elution
Tandem IPs
Date: 09/16/20
1. Weigh out 4 x 200mg of powder, add 800ul of extraction buffer per tube in safe-lock 2mL tubes.
2. Sonicate 5x2 sec @ 4 Amp and then repeat a second time (20 total sec, total J will be 25-30)
3. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
4. Combine clarified lysate from each of the tubes and then split evenly into 8 tubes
5. Set up 4x 200mg anti-FLAG IPs (using 10ul anti-FLAG beads per 100mg; 20ul beads for 200mg IP; this is based on Hua's recent titration of beads with this cell line 07/10/19 - LD401_anti-FLAG and anti-ORF1 beads titration)
6. Incubate @ 4°C for 30’
7. Prepare 1mg/ml 3x Flag peptide working sol'n: dilute 5mg/ml stock 1:5 in extraction buffer (20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 + PI + RNAsin)
8. After IP, wash beads with 2x 1ml extraction buffer Transfer beads to new Eppendorf tube after 2nd wash
9. Wash beads once more with 1ml extraction buffer
10. Add 50ul of 1mg/mL 3xFLAG to each tube containing anti-FLAG beads (50ul per 200mg IP)
11. Incubate @ RT for 15' with shaking.
12. Transfer the elution to a fresh tube.
13. Save the anti-FLAG beads and put them on ice. These will be eluted a second time with 1.1X LDS, but this can be done at the end.
14. Split the 3x FLAG elution (200ul total) into two aliquots:
Save 1/8 (25uL, 100mg) of the 3x FLAG elution, this will be the “Input” fraction
The other 7/8 (7x25=175uL, 700mg goes to anti-ORF1 tandem IP using 35uL anti-ORF1 beads (5ul anti-ORF1 beads per 100mg IP) split across 3 tubes (~60ul 3xFLAG elution and 12uL beads per tube for 233mg IP)
15. Incubate 3x FLAG elution with anti-ORF1 beads @ RT for 15’ with mixing
16. Save the supernatant after IP; this will be the “Sup” or “FT” (175ul; 25ul per 100mg)
17. Wash the beads in each tube with 1x 1ml of wash buffer
18. Elute in 10uL ORF1 di-peptide diluted with 1.5vol of 500mM buffer with no detergent to ~1mM (with protease inhibitors [no EDTA]) (P194712, B26, purity 91%, MW 2864; 2.5mM in 50mM HEPES, pH7.4, 500mM NaCl and 0.5% Triton); the final concentration of Triton will be 0.2%
19. Incubate @ RT for 15’ with mixing
20. Pool the elution per 3 tubes and put them on ice (30ul total)
21. Repeat elution step with 5uL buffer and add the elutions to the matching pooled elutions from the previous step (~15uL X 3 = 45uL total for 700mg), this will be “E” fraction. Save 9 ul of combined elution for gel analysis (6.4ul per 100mg)
22. Save the anti-ORF1 beads and put them at -20. These will be eluted a second time with 1.1X LDS, but this can be done at the end.
23. Save the anti-ORF1 beads after peptide elution and put them on ice. These will be eluted a second time with 1.1X LDS, but this can be done at the end.
Removing ORF1 di-peptide using Zeba 40K column
1. Remove the storage buffer from Zeba 40K column, spin @ 1000g for 1’
2. Equilibrate three 75ul Zeba 40K column (sample range: 3-12ul) with 3x 50ul of 20mM HEPES, pH 7.4, 300mM NaCl, NO DETERGENT, spin @ 1000g for 1’
3. Load 12ul of sample on column and spin @ 1000g for 1’ (manufacturer recommendation: spin 1000g for 2’); Only recovered 9ul for each column
4. Combine sample from 3 columns; save 8ul for gel; this will be “E_Zeba” (should have ~20ul left for EM). All 8ul was given to Igor for EM this time; no more for gel
5. Spin @ 1000g for another 1’, check the volume recovered (no extra volume this time)
LDS wash of the beads
Will check what remains on the beads after native elution and also do a side by side comparison of LDS wash at RT and 70C
anti-FLAG beads (80ul total for 800mg IP)
1. Add 250ul of 1xPBS to each tube of anti-FLAG beads (20ul for 200mg IP; 4 tubes)
2. Combine all beads and then split into 2 equal aliquots (40ul each
3. Add 40ul of 1.1x LDS to each tube
4. Elute one aliquot of beads @ RT for 10’ with mixing; Take 10ul for gel “LDS_anti-FLAG_RT” (100mg equivalent)
5. Elute the other aliquot of beads @ 70C for 5’ with mixing; “LDS_anti-FLAG_70C”; Take 10ul for gel “LDS_anti-FLAG_RT” (100mg equivalent)
anti-ORF1 beads (36ul total for 700mg IP)
6. Add 250ul of 1xPBS to each tube of anti-ORF1 beads (36ul for 700mg IP; 3 tubes)
7. Combine all beads and then split into 2 equal aliquots
8. Add 35ul of 1.1x LDS to each tube
9. Elute one aliquot of beads @ RT for 10’ with mixing; Take 10ul for gel “LDS_anti-ORF1_RT” (100mg equivalent)
10. Elute the other aliquot of beads @ 70C for 5’ with mixing; “LDS_anti-FLAG_70C”; Take 10ul for gel “LDS_anti-ORF1_RT” (100mg equivalent)
Sypro gel to check all fractions
1. Unstained Marker_1ul
2. Input (3xFLAG elution)_25ul (100mg)
3. Sup (FT of anti-ORF1 IP)_25ul (100mg)
4. E_Zeba (E in 300mM buffer)_6.4ul (100mg)
5. LDS_anti-FLAG_RT_10ul
6. LDS_anti-FLAG_70C_10ul
7. LDS_anti-ORF1_RT_10ul
8. LDS_anti-ORF1_70C_10ul
9. BSA_100ng
10. Prestained Marker_5ul
