LINE-1 tandem prep_ORF1p_nanbody_test for EM
Cell line pLD401 (Light)
Completed in “RNA style” using nuclease-free reagents
Scale: 1000mg (5x 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 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
Ref: 09/30/20 (previously done on 9/16/20) Wiki entry: September 30, 2020 - LD401 Tandem for EM
1. Weigh out 5 x 200mg of powder (total 1g), add 800ul of extraction buffer per tube in safe-lock 2mL tubes.
2. Sonicate 5x2 sec @ 2 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 5x 200mg anti-FLAG IPs (use 20ul beads for 200mg IP; 100ul 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 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. Add 30ul of extraction buffer to each tube to wash the beads and capture any residue eluate trapped in beads
14. Combine wash with elution (5x 50 + 5x 30 = 400uL 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 2 tubes (~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; Treat beads in each tube separately after this step
20. Tube a: 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 (15ul total)
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), this will be “E” fraction for tube a (“Ea”. 24ul total, 4.8uL per 100mg). All 24ul will be desalted
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 b: Incubate beads with 10ug of ORF1p nanobody (LaORF1-5) @ RT for 15’ with mixing
● LaORF1-5: 1.25mg/ml, in 20mM HEPEs, pH7.4, 150mM NaCl. Take 10ul (10ug) of nanobody, add 15ul of 20mM HEPEs, pH7.4, 500mM NaCl (25ul @ 0.5ug/ul; Spike 20ul to anti-ORF1 beads; save the rest for gel analysis)
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, “Eb” (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 (2 for Ea and 2 for Eb) on column and spin @ 1500g for 2’
4. Combine same sample from 2 columns; save 4ul for gel (80mg equivalent); this will be “Ea_300” and “Eb_300” (Desalted sample in 300mM buffer; should have ~20ul left for EM)
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 (20ul for 200mg IP; 5 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; 2 tubes)
5. Keep anti-ORF1 beads for a and b 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 and b” 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 0.5ug/ul = 250ng) and after binding to anti-ORF1 beads; run these two samples side by side (Take 1ul of nanobody before and after binding, diluted with 8ul of 1.1x LDS, add 1ul of 0.5M DTT; load half)
Sypro gel to check all fractions
1) Unstained Marker_1ul
2) Input_10mg
3) FT_10mg
4) Ea_300 (- nanobody, after desalting)_4ul (80mg)
5) Eb_300 (+ nanobody, after desalting)_ 4ul (80mg)
6) Space
7) LDS_anti-FLAG_beads_8ul (80mg)
8) LDS_anti-ORF1-a_beads_8ul (80mg)
9) LDS_anti-ORF1-b_beads_8ul (80mg)
10) Space
11) Nanobody_before binding (250ng; 0.5ul)
12) Nanobody_after binding (0.5ul)
13) BSA_50ng
14) BSA_100ng
15) Prestained Marker_5ul
