Protein Purification (ProA - tagged constructs)
Supplies:
Dynal Biotech (phone number: 1-800-638-9416)
Dynabeads M-280 Tosylactivated (product number 142.03)
Magnetic Particle Concentrator (product number: 120.01)
Ideal for 1.5 ml Eppindorf tubes to 50 ml Falcon tubes
Rabbit Gamma Globulin (product number 011-000-002)
Buffer A: 0.1 M
Na-phosphate buffer pH 7.4
Buffer C: PBS pH 7.4
(phosphate buffered saline) with 0.1% (w/v) BSA
Buffer D: 0.2 M Tris
pH 8.5 with 0.1% (w/v) BSA
IP Lysis Buffer: 50mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton
IgG Coupling:
a. Resuspend
Dynabeads M-280 Tosylactivated well by pipetting and vortexing for
approximately 1 min. Avoid foaming.
b. Immediately pipette the volume
to be used into the desired test tube.
c. Place the tube in a magnet
(Dynal MPC) for 1-4 minutes.
d. Pipette off the supernatant
carefully, leaving beads undisturbed.
e. Remove the test tube from the
Dynal MPC and resuspend the beads carefully in an ample volume of Buffer
A. Mix gently for 2 min.
f. After using the magnet again
and pipetting off the supernatant, resuspend the washed beads in the same
volume of Buffer A as taken from the tube in point d, or to the desired
concentration.
g. The Dynabeads M-280
Tosylactivated are now washed and ready for coating.
h. Make a
homogeneous suspension of the Dynabeads M-280 Tosylactivated by using a pipette
and by vortexing for approximately 1 min. Pipette out the desired number of
beads and wash as previously described.
i. The rabbit IgG comes in
Buffer A, therefore buffer exchanging will not be necessary. For coupling, the concentration is ~ 2mg IgG
/ ml beads, but add a bit more for good measure.
j. Again, resuspend the
Dynabeads thoroughly (remove tube from the magnet and vortex or use
ultrasound), add rabbit IgG and continue to vortex for 1 minute. We recommend a
concentration of 1-2 x 109 Dynabeads per ml final coating solution.
k. Incubate for 16-24 h at 37°C
with slow tilt rotation.
l. After incubation,
place the tube in the magnet for 1 - 4 minutes and remove the supernatant.
m. Wash the coated
beads four times
- 2x in Buffer C for 5 minutes
at +4°C
- 1x in Buffer D for 24 h at
+20°C or for 4 h at 37°C. (Tris will block free tosyl-groups)
- 1x in Buffer C for 5 minutes
at +4°C.
n. The Dynabeads M-280
Tosylactivated are now coated and ready for use.
a. Grow cells overnight to OD 1-2 (closer to 1
is ideal)
b. Spin cells at 4000 rpm for 10 minutes and
aspirate media
c. Resuspend cells in either PBS or STOP
buffer. Spin down and pellet
Method 1: Bead Beater
d. Resuspend cells in IP lysis buffer + protease
inhibitors. Try to keep the volume low
at this point – you can always add more later.
I usually add about 15 mls for a 20 ml pellet to start.
e. Add mixture into large bead beater container with glass lysis beads. This should be pre-cooled in freezer. The container should be 50% full of beads and have about ¾ of an inch of liquid on top. The idea is during lysis to make sure that you see a nice vortex. This is not always easy and may require some tricks like manually tilting the apparatus or adding more buffer or beads.
f. During lysis, it is critical to keep chilled. It will heat up!!
- keep lysis time short 30 s to 1.5 min.
- allow several minutes in between run times
- add dry ice to the outer chamber.
- perform in cold room.
h. Cell lysis can take up to one hour. Take samples about every 3rd time to check lysis under light microscope. You can expect between 60-80% cell breakage.
i. When finished, extract lysate using a 5 ml plastic pipette. Be very careful when drawing off lysate not to pick up excess glass beads. This can be avoided by pushing nozzle to the bottom and drawing up the lysate. Beads should be rinsed 2-3 times with fresh buffer.
j. Lysate should be put into pre-chilled plastic centrifuge tube and spun for 20 minutes @ 16K rpm.
k. Collect supernatant for analysis.
Method 2: Mortar and Pestle Lysis
d. Cells should be scooped out and placed into a pre-chilled pestle. The pestle should be sitting on top of dry ice. Remember, if your cells aren’t completely frozen, this method will not work.
e. Scoop liquid nitrogen on top of cells to flash freeze. This will have to be done intermittingly during lysis.
f. Start smashing. Cells will fall out of pestle. I try to avoid this by placing a piece of cardboard on top with a hole in the middle.
g. Cells should be ground to a fine powder. You can check under the microscope. You can expect between 50-60% cell breakage.
h. Resupend powder in IP Lysis buffer and proceed from J of Method 1.
a. Add beads to lysate. I usually add 100 ml per 2 liter culture. Rotate cells at 4C for 3-5 hours. I usually use 50 ml falcon tube for the purification.
b. Use your Magnetic Particle Concentrator to isolate beads. This takes a couple of minutes if you are using a 50 ml falcon tube. Place in a clean 15 ml Falcon tube.
c. Wash 4 times with IP lysis buffer, each time concentrating and washing. I try to avoid strong vortexing for the wash steps.
d. Resuspend beads in original volume in an Eppindorf tube. If you use originally used 500ml of beads, then concentrate in ~ 1ml of buffer.
e. Pull out 1% of mixture for Western Blot analysis. Concentrate beads using the Magnetic Particle Concentrator.
f. Elute beads with 1% SDS in desired volume. For 500ml of beads, use ~ 200ml. Gently vortex for ~20-30 seconds. Concentrate beads and extract supernatant. Extract 1% sample for Western Blot Analysis.
g. Resuspend beads in ~ 1ml of lysis buffer. See step e. Pull out 1% of mixture for Western Blot analysis.
h. Analyze samples. I always check binding efficiency (pre, post binding, beads), elution efficiency (steps e, f, g) as well as running 5-10% of the supernatant on a small gel and silver staining to determine how much and how clean the purification is.
For any questions:
Email Rory Geyer: