LINE-1 tandem prep with or without nanobody for EM
Date: 06/09/21
Cell line pLD401 (light)
Completed in “RNA style” using nuclease-free reagents
Scale: 6x 500mg IPs
Extraction buffer: 20mM HEPES, pH7.4, 1% Triton X-100, 500mM NaCl + PI + RNase Inhibitor @ 1:250 in extraction buffer (1:1000 in wash buffer)
Native elution at 300 or 500mM NaCl - detergent concentration lowered to 0.12%
Tandem IPs
Reference: LD401 Tandem IP for EM_02/10/21 Wiki entries: February 10, 2021 - LD401 Tandem IP for EM_test ORF1 nanobody binding; March 24, 2021 - LINE-1 tandem prep_with or without LaORF1 for EM
1. Weigh out 12 x 250mg of powder (total 3g), add 1000ul of extraction buffer per tube in safe-lock 2mL tubes.
2. Sonicate 5x 2 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 (6x 500mg IPs)
Anti-FLAG IP
5. Set up 6x 500mg anti-FLAG IPs (use 50ul beads for 500mg IP; 300ul 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 750ul 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 125ul of 1mg/mL 3xFLAG to each tube containing anti-FLAG beads (we used 50ul per 200mg IP previously; need to increase this to 125ul for 500mg IP)
11. Incubate @ RT for 15' with shaking
12. Transfer the elution to a fresh tube
13. Add 75ul 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; 75ul for 500mg)
14. Combine wash with elution (6x 125 + 6x 75 = 1200uL total)
15. Save the anti-FLAG beads; wash beads with 1ml 1x PBS and store beads in storage buffer @ -20C.
Anti-ORF1 IP
16. Save a small aliquot of 3x FLAG elution for gel; use the rest for anti-ORF1 IP • Save 4ul (1200ul for 3g; 4ul for 10mg equivalent) of the 3x FLAG elution, this will be the “Input” fraction
• Split the rest evenly across 3 tubes (~1g each) for anti-ORF1 tandem IP; using 50ul anti-ORF1 beads per tube (5ul anti-ORF1 beads per 100mg IP)
• Tubes 1-3 will subject to different wash steps before native elution and also salt concentration will be different during native elution
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. Take 2x 50ul of FT and dialyze the samples to get rid of detergent (use 7K and 20K Slide-A-Lyzer Mini Dialysis Units from ThermoFisher
20. Snap freeze the rest and stored at -80C.
Dialyze anti-ORF1 IP FT using Slized-A-Lyzer Mini dialysis units from ThermoFisher (7K, Prod.# 59560; 20K MWCO, Prod. # 69590)
Place the unit into the float so that the bottom of the dialysis unit is in contact with the dialysate,Always make sure that the volume level of the sample is at or above the level of the dialysate.
To prevent contamination, do not touch the membrane with ungloved hands
If glycerol removal is desired, soak the unit in 1L of water for 15 minutes
Load 50ul of FT to each unit (one 7K and the other one 20K) (for best result, sample volume should be 10-100ul)
Cap the unit and place in a flotation device (Prod. # 69588)
Use a low-speed setting on a stir plate so that the flotation device is not submerged
Typical dialysis time to obtain equilibrium is 10 minutes to 2 hours using a dialysate volume of 0.5-1L
For the best volume recovery, collect the sample from the corner of the unit
Save 4ul from each unit (7K and 20K) for gel
Make new 2.5mM stock with 5mM CHAPS (0.3%)
ORF1 dipeptide from 21st Century (average MW of dipeptide is 2865)
50mM HEPEs, pH 7.4, 500mM NaCl, 5mM CHAPs (When using 20mM HEPEs, the pH of peptide solution is lower than 6 and the peptide will not dissolve)
Take one vial (5mg @ 98% purity)
2.5M => 2.5x 2865mg/ml; 2.5mM => 2.5x 2865ug/ml=7.162mg/ml To make 2.5mM stock, 5mg should be dissolved in 0.698ml of buffer with 5mM CHAPs
I. Elute in 500mM as we normally do, and then dilute to 300mM
These samples could be used for both direct dilution (tube 1) and micro-dialysis (tube 2).
21. Tube 1: Wash beads with 3x 1ml extraction buffer
22. Tube 2: Wash beads with 2x 1ml extraction buffer followed by 1x 1ml of 20mM HEPEs, 500mM NaCl; 2mM CHAPS
23. Tube 1 (direct dilution): Elute in 36ul 1mM ORF1 di-peptide with 500mM salt, 2mM CHAPS Dilute 2.5mM stock in 50mM HEPES, pH7.4, 500mM NaCl and 5mM CHAPS with 1.5vol of 500mM buffer with no detergent to 1mM with protease inhibitors; final concentration 500mM NaCl, 2mM CHAPS
24. Tubes 2 (for dialysis): Elute in 36ul 1mM ORF1 di-peptide with 500mM salt with 0.2% Triton (Old stock)
Dilute 2.5mM stock in 50mM HEPES, pH7.4, 500mM NaCl and 0.5% Triton with 1.5vol of 500mM buffer with no detergent to 1mM with protease inhibitors; final concentration 500mM NaCl, 0.2% Triton X-100
25. Incubate @ RT for 15’ with mixing
26. Collect the eluate and keep it on ice (36ul)
27. Tube 1 (direct dilution): wash beads with 24ul 20mM HEPEs; add it to elution from previous step
Final salt and detergent concentrations: 300mM NaCl; 1.2mM CHAPS (0.07%)
Final volume: 60ul (60ul total for 1g; 6ul per 100mg).
28. Tube 2 (for dialysis; elution step will be the same as previous experiment 03/24/21): wash beads with 24ul of 20mM HEPEs, 300mM NaCl, no detergent (same as dialysis buffer) and add it to elution from the previous step Final salt and detergent concentrations: 420mM NaCl; 0.12% Triton X-100 Final volume: 60ul (60ul total for 1g; 6ul per 100mg).
29. 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.
II. During the washing step we do the first wash with 500, second wash with 400, 3rd wash with 300 and elute at 300
This sample seems fine for direct spotting (tube 3)
30. Wash beads with 1x 1ml of 20mM HEPEs, 500mM NaCl; 1% Triton X-100
31. Wash beads with 1x 1ml of 20mM HEPEs, 400mM NaCl; 1% Triton X-100
32. Wash beads with 1x 1ml of 20mM HEPEs, 300mM NaCl; 2mM CHAPs
33. Tubes 3 (direct spotting): Elute in 36ul 1mM ORF1 di-peptide with 300mM salt with 2mM CHAPS
Dilute 2.5mM stock in 50mM HEPES, pH7.4, 500mM NaCl and 5mM CHAPS with 1.5vol of 167mM buffer with no detergent to 1mM with protease inhibitors; final concentration 300mM NaCl, 2mM CHAPS
34. Wash beads with 24ul of 20mM HEPEs, 300mM NaCl, no detergent; add it to elution from previous step
Final concentration of peptide elution: 300mM NaCl; 1.2mM CHAPS (0.07%)
Final volume: 60ul (60ul total for 1g; 6ul per 100mg).
35. Save the anti-ORF1 beads; wash beads with 1ml 1x PBS and store beads in storage buffer @ -20C.
36. Save 5% of elution for gel (50mg scale, 5% x 60ul = 3ul; 57ul left)
At this point, we have 3 tubes of native elution from 1g tandem IP
● Tube 1: 500mM peptide elution -> direct dilution
● Tube 2: 500mM peptide elution -> dialysis
● Tube 3: 300mM peptide elution
37. Split the rest of sample in Tubes 1 and 3 into 2 equal aliquots (“a” without nanobody and “b” with 5ng of nanobody added to the sample)
There will be ~28ul (57ul/2) in each tube
Introducing ORF1p nanobody after native elution and before dialysis
Prepare 3x 5ug of ORF1 nanobody (LaORF1-5) with no detergent
● LaORF1-5 stock: 1.25mg/ml, in 20mM HEPEs, pH7.4, 150mM NaCl.
● Take 12ul of stock (12x1.25ug/ul), spin @ 20k rcf, 4°C for 10’ and keep the supernatant (get rid of any potential nanobody aggregate)
● Take supernatant, add 4ul (5ug) into each ORF1 peptide elution sample Tube 1 (direct dilution) and tube 3 (300mM peptide elution): spike in nanobody and use sample for EM directly Tube 2 (500mM peptide elution): spike in nanobody and dialyze the sample in 300mM buffer before using the sample for EM; see below
Tube 2: Buffer exchange using micro Tube-O-Dialyzer (4K MWCO G-Biosciences, cat # 786-611)
PREPARATION BEFORE USE
Tube‐O‐DIALYZERTM are supplied in a preservative to maintain quality. Prior to use discard the preservative from the tube and place the dialysis cap upside down in a beaker or other suitable container and add 1‐2ml DI water or dialysis buffer to rinse. Keep the Tube‐O‐DIALYZERTM membrane wet until required
INSTRUCTIONS FOR USE
1) Pipette sample directly into the Tube‐O‐DIALYZERTM tube. For Tube‐O‐ DIALYZERTM Micro use 20‐250μl.
a. Add 5ng of nanobody to Tube 2b
b. Save 10% for gel from tube 2b (3ul)
c. Load the rest in dialysis tube (~28ul in 2a and ~29ul in 2b)
2) Remove the Tube‐O‐DIALYZERTM dialysis cap from the rinse water/buffer and carefully remove excess liquid with a pipette tip.
3) Screw the dialysis cap on to the Tube‐O‐DIALYZERTM tube until finger tight. Invert the Tube‐O‐DIALYZERTM, ensuring the entire sample rests upon the membrane.
4) Keeping the Tube‐O‐DIALYZERTM in an inverted position, slide the supplied float onto the Tube‐O‐DIALYZERTM tube. Place the Tube‐O‐DIALYZERTM in the dialysis buffer.
5) Ensure that the dialysis membrane contacts the dialysis buffer. If there are large air a. bubbles trapped underneath the dialysis membrane surface, tilt the tube or squirt buffer to remove the air bubbles. Gently, stir the dialysis buffer. For efficient and complete dialysis we recommend inverting or gently tapping the Tube‐O‐ DIALYZERTM 1‐2 times during dialysis to mix the sample. If necessary repeat the centrifugation in step 3.
6) Dialysis Time: Dialysis time will depend on the nature of sample, MWCO of the Tube‐O‐DIALYZERTM, sample and dialysis buffer volume and concentration. Higher MWCO will allow faster dialysis. As a guide, the sample should be dialyzed for 2‐ 12h. Dialysis buffer should also be replaced at least once during dialysis.
7) After dialysis, remove the Tube‐O‐DIALYZERTM from the float and immediately spin the Tube‐O‐DIALYZERTM (in up‐right position) for 5‐6 seconds at 500‐1,000xg. NOTE: Do not spin longer as this may cause the membrane to rupture.
8) Discard the dialysis cap and replace with the supplied Storage Cap.
Note: When loading the dialysis unit, part of 2a (-nanobody) got mixed with 2b (+ nanobody). So, 2a volume was reduced (13ul after dialysis) and 2b volume was increased (45ul after dialysis). Take 3ul from each for gel.
Another mistake: 4x LDS was added to 10ul of leftover 2a (supposed to be for EM), not the 3ul aliquot for gel. Take 30% of the 2a/LDS mix. Save the 3ul of 1a for EM.
Final buffer conditions of EM samples:
1a and 1b -> direct dilution: 300mM NaCl; 1.2mM CHAPS
2a and 2b -> dialysis: 300mM NaCl; no detergent
3a and 3b -> direct dilution: 300mM NaCl; 1.2mM CHAPS
Sup containing ORF2p (FT 7K and FT 20K) -> dialysis: 300mM NaCl; no detergent
LDS wash of the beads
Try to see what is left on anti-FLAG beads and compare the efficiency of native elution of anti-ORF1 IP
Elution conditions of anti-ORF1 beads are:
Tube 1 (1a and 1b): 500mM NaCl, 2mM CHAPS
Tube 2 (2a and 2b): 500mM NaCl; 0.2% Triton X-100
Tube 3 (3a and 3b): 300mM NaCl, 2mM CHAPS
Take 1 tubes of anti-FLAG (for 500mg IP) or Tubes 1, 2 and 3 of anti-ORF1 (for 1g IP)
Wash with 1ml of 1x PBS
Resuspend beads in 1ml of 1x PBS
Take 50ul of resuspension (25mg for anti-FLAG and 50mg for anti-ORF1)
Sypro gel to check all fractions
Add LDS and DTT to each sample (final concentration 1x LDS and 50mM DTT)
Heat samples @ 70°C for 10’
Load 26-well 4-12% Bis-Tris gel
1) Unstained Marker_1ul
2) Space
3) Input_10mg
4) FT_before dialysis_10mg
5) FT_after dialysis (7K) _10mg
6) FT_after dialysis (20K)_10mg
7) 1a_direct dilution- nanobody_3ul
8) 1b_direct dilution+nanobody_3ul (4ul nanobody added to 28.5ul elution, ignore the 15% volume increase here)
9) 2a_dialysis-nanobody_before_3ul
10) 2a_dialysis-nanobody_after_3ul
11) 2b_dialysis+nanobody_before dialysis_3ul
12) 2b_dialysis+nanobody_after dialysis_3ul
13) 3a_direct spotting-nanobody_3ul
14) 3b_direct spotting+nanobody_3ul
15) BSA_10ng
16) BSA_50ng
17) BSA_100ng
18) Prestained Marker_0.2ul
19) LDS_anti-FLAG beads (beads for 25mg IP)
20) LDS_anti-ORF1 beads-1_direct dilution (beads for 50mg IP)
21) LDS_anti-ORF1 beads-2_dialysis (beads for 50mg IP)
22) LDS_anti-ORF1 beads-3_direct spotting (beads for 50mg IP)
23) Space
24) Space
25) 2.5ul of Prestained marker (not shown)
