LINE-1 tandem prep_with or without LaORF1 for EM
Date: 03/24/21
Cell line pLD401
Completed in “RNA style” using nuclease-free reagents
Scale: 3x 500mg (6x 250mg IPs)
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 lowered to 0.2%
• 100mg of powder yields an estimated ~500ng of total protein via native elution (This estimation was from Angelicas Wiki entry)
Tandem IPs
Reference: LD401 Tandem IP for EM_02/10/21 Wiki entry: February 10, 2021 - LD401 Tandem IP for EM_test ORF1 nanobody binding
1. Weigh out 6 x 250mg of powder (total 1.5g), add 1000ul of extraction buffer per tube in safe-lock 2mL tubes. (3x 500mg at the end; a: normal control with desalting; b: nanobody added after desalting; c: nanobody added before native elution)
2. Sonicate 5x2 sec @ 2 Amp and then repeat a second time (20 total sec, total energy ~30J); Since we have 250mg here, double check to make sure that the powder is fully resuspended
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 6 tubes]
5. Set up 6x 250mg anti-FLAG IPs (use 25ul beads for 250mg IP; 150ul beads total)
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) (will need 250ul total for native elution)
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 62.5ul of 1mg/mL 3xFLAG to each tube containing anti-FLAG beads (we used 50ul per 200mg IP previously; for 250mg per IP, need to increase this to 62.5ul)
11. Incubate @ RT for 15' with shaking
12. Transfer the elution to a fresh tube
13. Add 37.5ul of extraction buffer to each tube to wash the beads and capture any residue eluate trapped in beads (we used 30ul per 200mg IP previously; for 250mg per IP, need to increase this to 37.5.5ul)
14. Combine wash with elution (6x 62.5 + 6x 37.5 = 600uL total)
15. 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
16. Save a small aliquot for Western; use the rest for anti-ORF1 IP
• Save 4ul (1%, 10mg equivalent) of the 3x FLAG elution, this will be the “Input” fraction
• Split the rest evenly across 3 tubes (Tubes a, b and c) (~500mg each) for anti-ORF1 tandem IP; using 25uL anti-ORF1 beads per tube (5ul anti-ORF1 beads per 100mg IP)
17. Incubate 3x FLAG elution with anti-ORF1 beads @ RT for 15’ with mixing
18. Save the supernatant after IP; this will be “FT” (200uL per 500mg IP; load 4ul for 10mg)
19. Wash the beads in each tube with 1x 1ml of wash buffer
Tubes a, b and c will be treated differently when subject to LaORF1 nanobody (starting at steps 20 and 25)
Introducing ORF1p nanobody before and after desalting
We want to estimate the molar quantity of ORF1p from the gel staining intensity - then spike in 2:1 nanobody (avoiding massive excess) - then EM image. (Will use 5ug of nanobody instead of 10ug used in previous experiment 02/10/21)
This was assessed / estimated in the following way:
• By eyeballing the gel, ~25% (2.5ug) of the 10ug input was estimated to be depleted after incubation with the sample. Therefore, total nanobody could be reduced to 5ug.
o After the last incubation, the remaining nanobody was discarded. This should be SAVED.
• By eyeballing the gel, ORF1p was estimated at 100ng (compared to BSA 100ng) and this was known to be 2% of the total samples loaded, making total ORF1p ~5ug. Anti-ORF1p nanobody (at ~15kDa) is ~1/2 the mass of ORF1p (40kDa), therefore, 5ug of nanobody should represent ~2:1 molar ration to ORF1p.
Prepare 2 aliquots of ORF1 nanobody; one with detergent (will be used for binding; Tube b) and the other one without detergent (will be used after desalting)
● LaORF1-5: 1.25mg/ml, in 20mM HEPEs, pH7.4, 150mM NaCl. Take 20ul (10ug) of
● LaORF1+Tx: 5ul of 1.25mg/ml (6.25ug), in 20mM HEPEs, pH7.4, 150mM NaCl. Take 5ul (6.25ug) of nanobody, add 20ul of 20mM HEPES, pH7.4, 588mM NaCl, 0.25% Triton X-100 (25ul @ 0.25ug/ul; final 500mM NaCl, 0.2% Tx); Spike 20ul (5ug) to anti-ORF1 beads; save the rest for gel analysis) …
● LaORF1-Tx: 5ul of 1.25mg/ml (6.25ug), in 20mM HEPEs, pH7.4, 150mM NaCl. Take 5ul (6.25ug) of nanobody, add 1.25ul of 20mM HEPEs, pH7.4, 900mM NaCl (6.25ul @ 1ug/ul; final 300mM NaCl, no detergent); Spike 5ul (5ug) to anti-ORF1 beads; save the rest
● After diluting the nanobody, spin @ 20k rcf, 4°C for 10’ and keep the supernatant (get rid of any potential nanobody aggregate)
20. Tube a and b: Elute in 18uL 1mM ORF1 di-peptide (dilute 2.5mM stock 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%
21. Incubate @ RT for 15’ with mixing
22. Collect the eluate and keep it on ice (18ul)
23. Repeat elution step with 6uL of 20mM HEPEs, 300mM NaCl, no detergent (same as buffer for desalting) and add it to the elution from the previous step (24uL total for 500mg; 4.8uL per 100mg). “a” and “b” will both undergo desalting. Will add LaORF1 nanobody to “b” after desalting; “a” will be used for EM directly after desalting
24. 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.
25. Tube c: Incubate beads with 5ug of ORF1p nanobody (20ul of LaORF1-5 + Tx) @ RT for 15’ with mixing
26. Collect the nanobody solution after binding; will compare it with nanobody before binding to check the depletion of the nanobody
27. Wash beads with 1ml of extraction buffer; then repeat steps 23-24 and collect elution fraction for tube b, “Ec” (18ul ORF1 peptide elution + 6ul wash = 24ul total for 500mg)
Removing ORF1 di-peptide using Zeba 40K column
1. Remove the storage buffer from Zeba 40K column, spin @ 1500g for 1’
2. Equilibrate four 75uL Zeba 40K column (sample range: 3-12ul) with 3x 50ul of 20mM HEPES, pH 7.4, 300mM NaCl, NO DETERGENT, spin @ 1500g for 1’
3. Load 12ul of sample (1 for each: Ea, Eb and Ec; 6 columns total) on column and spin @ 1500g for 2’; half of Ea was saved without desalting (can always pass it through the desalting column if needed)
4. Combine same sample from 2 columns; save 4ul for gel (80mg equivalent); this will be “Ea_300”, “Eb_300” and “Ec_300” (Desalted sample in 300mM buffer)
5. After desalting, add 5ul of LaORF1-Tx to Ea_300 (24ul + 5ul = 29ul total)
LDS wash of the beads
Will check what remains on the beads after native elution by LDS wash at RT
1. Add 250ul of 1xPBS to each tube of anti-FLAG beads (25ul for 250mg IP; 6 tubes)
2. Combine all anti-FLAG beads; Take 10% of the beads, add 10ul of 1.1x LDS; keep the rest @ 4°C
4. Add 250ul of 1xPBS to each tube of anti-ORF1 beads (25ul for 500mg IP; 3 tubes)
5. Keep anti-ORF1 beads for a, b and separately; Take 20% of the beads from each tube, add 10ul of 1.1x LDS; Keep the rest @ 4°C
6. Elute beads @ RT for 10’ with mixing
7. Collect LDS wash (10ul); “LDS_anti-FLAG” and “LDS_anti-ORF1-a, b and c” beads Load 8 ul (80mg scale)
Prepare gel samples of the nanobody before and after binding
Take 0.5ul of nanobody before (0.5ul x 0.25ug/ul = 125ng) and after binding to anti-ORF1 beads; run these two samples side by side
Sypro gel to check all fractions
1) Unstained Marker_1ul
2) Input_10mg (10mg out of 500mg per tube; 2%)
3) FT_10mg
4) Ea_Un-desalted_4ul
5) Ea_300 (desalted; normal control, no nanobody at all)_4ul (80mg)
6) Eb_300 (desalted; with nanobody added after desalting)_4.8ul (80mg; total volume increased from 24 to 29ul after adding 5ul of nanobody)
7) Ec_300 (desalted; with nanobody added before native elution)_ 4ul (80mg)
8) LDS_anti-FLAG_beads_8ul (80mg)
9) LDS_anti-ORF1-a_beads_8ul (80mg)
10) LDS_anti-ORF1-b_beads_8ul (80mg)
11) LDS_anti-ORF1-c_beads_8ul (80mg)
12) LaORF1 + Tx_before binding (125ng; 0.25ul out of 20ul, 2.5%)
13) LaORF1 + Tx_after binding (0.5ul)
14) BSA_100ng
15) BSA_50ng + Prestained Marker_5ul
