Isolation of HA-TEV conjugated protein (3 gram scale)
(modified from HA-TEV-Arp prep by JH)
1. Generate cross-linked anti-HA antibody-coated beads (CL4B sepharose), following
the Goode lab protocol. These are stored at 4°C in TBST buffer with azide, usually as
a 1:1 slurry (beads to buffer).
2. Remove the amount of beads you will need for this preparation into a 1.5ml tube (use
30µl beads per 3g lysate). Note: 30µl beads = 60µl of a 1:1 slurry. Important: USE a
P200 and cut-off yellow tip, pipette up and down to suspend the bead slurry evenly
before removing the 60µl.
3. Wash beads 2 x 1.5ml HEK buffer [20mM Hepes, pH 7.5, 1mM EDTA, 50mM
EDTA].
4. To generate a yeast high speed supernatant (HSS), use a thin spatula to weigh 3g
lysed yeast powder (-80°C) into a 15 ml tube (zeroed on the scale).
5. Add 3ml room temperature HEK buffer and begin thawing by heat of hand and
inverting the tube.
6. When the mixture is thawed, but still cold, add 30µl PMSF (0.2M stock) and mix by
inverting several times. Put tube back on ice.
7. Using a P1000, transfer lysate to 2 TLA100.3 tubes on ice.
8. Centrifuge 20 min., 80,000 rpm, 4°C, TLA100.3 rotor.
9. Use a P1000 to remove HSS into 15ml tube on ice. Carefully avoid the upper lipid
phase and the lower detritus. You should recover about 4ml of HSS.
10. Transfer 1.7ml HSS to each of two 1.5ml tubes on ice. Also, transfer 100µl of HSS to
a 1.5ml tube for a gel sample (called “S1”). To each 1.7ml HSS tube, add 15µl beads
(i.e. 30µl of 1:1 slurry).
11. Incubate the reactions in the cold room, rotating on a Labquake for approximately 1
hour (a couple hours longer is okay, but do not leave reactions over night).
12. Pellet beads in a low speed centrifuge, 3-4000 rpm, 20 sec. at room temperature.
Remove the HSS, saving 100µl in a 1.5ml tube on ice for making a sample (called
“S2”). Note (IMPORTANT): at this point, it is wise to combine the two reactions,
i.e., transfer the 15 + 15µl of beads into one tube (use a P200 and cut tip). This makes
all the washes below less work-intensive.
13. Wash beads 4 x 1.7ml cold HEK buffer. Each time, remove tube to a rack and rest 1520 sec. so the beads settle to the bottom (this makes it easier to remove the wash
supernatant w/o accidentally discarding some of the beads – value the beads!).
14. Wash beads 1 x 1.7ml HEK buffer + 0.5 M KCl. Make 10ml of this high salt wash
buffer by adding 2ml of 2.5M KCl to 8ml HEK buffer.
15. Wash beads one more time in 1.7ml HEK buffer alone.
16. Using a P1000, remove and discard most of the supernatant. Leave the 30µl of beads
with about 30µl buffer covering (i.e. 1:1 slurry). Transfer 10µl beads (20µl of 1:1
slurry) to a 1.5ml tube for a gel sample (called “P1”).
17. Add 1µl 6HIS-TEV protease (10 units/µl Invitrogen) to the remaining 40µl bead
slurry (1:1), and using a P200 and cut-off tip, gently pipette up and down to mix.
Save the tip for mixing (see below).
18. Incubate (without rotating) at room temperature (or 25°C bath) for 1-1.5 hr. At 30
min. intervals (i.e. once or twice during the reaction), use the tip from above to gently
pipette up and down, stirring up the beads.
19. Pellet beads and transfer supernatant to a new tube on ice. Use a P20 and beveled tip
to avoid taking up beads, while maximizing recovery of the supernatant (i.e. Get all
the liquid you can, but don’t take the beads). You should obtain about 20-30µl in your
tube.
20. Add 10µl to 5µl 3X SDS sample buffer (Important: No DTT or BME in sample
buffer!!!!) and boil for 1 min. (this is called the “P2” sample).
21. Prepare the remaining gel samples. Using a P200, transfer the S1 and S2 samples
(100µl) into tubes in a boiling water bath that contain 50µl of boiling 3X SDS sample
buffer. Boil 3-4 min., remove tubes to a rack at room temperature (not ice; things can
crash in SDS after boiling). If you are not loading on a gel within the next 30 min.,
store the tubes at –20°C, then boil samples for 1 min. before loading on a gel.
22. For the P1 sample, use a P20 to remove and discard the remaining buffer covering
beads. Add 10µl 1X sample buffer and boil beads for 1 min. Pellet beads and transfer
supernatant to a new 0.5ml tube. Load sample on gel or store as above.
23. Run a 12% SDS-PAGE gel. Blot S1 and S2 samples for HA, and analyze P1 and P2
samples by Coomassie staining. On one gel, load 6µl BMW standards, 6µl S1 and 6µl
S2. Blot these lanes (1:5000 anti-HA HRP-conjugated antibody, no secondary) and
develop by ECL. Skip a few lanes and load 6µl and 2µl BMW standards, then 1215µl P1 and P2 samples. Coomassie stain this portion, which roughly quantifies
yield; the BMW standards contain 0.6µg and 0.2µg protein per band, respectively.