1. AOT (Bis(2-ethylhexyl)sulfosuccinate sodium salt, Fluka #86139), 100 mg.
2. Toulene.
3. Fluorescent dextran (>=500 kDa), 20 mg/ml in H2O.
4. MeOH, 100 ml.
5. PBS.
6. Acrylamide (40%, Bio-Rad) and Bis (2%, Bio-Rad).
7. Ammonium persulfate (Bio-Rad) solution, 10 mg in 100 ul distilled water. Prepare immediately before use in step 10.
8. TEMED (Bio-Rad).
9. MES buffer, 0.1 M, pH 4.9, 10 ml.
10. EDC, 26 mg/ml, prepare immediately before use.
11. Protein for coating, 10 mg/ml.
12. Acrylic acid (Fluka), comes as liquid.
1. Fit a 15 ml corex tube with a rubber stopper with a yellow pipette tip inserted through a hole. Place a flea size stir bar in the tube and clamp onto a ring stand in a hood. Set up a stirring plate under the tube and connect the stopper to the source of nitrogen gas.
2. Tare the corex tube w/o stopper and add AOT 40-60 mg. Add toulene to make a final concentration of 10 mg/ml AOT. Stir to dissolve.
3. Mix 5 ml of acrylamide solution in a small beaker according to the dilution scheme below.
| Final Acryl/Bis | 40%Acrylamide | 2%Bis | 1M HEPES | H20+Other | Young's Modulus |
|---|---|---|---|---|---|
| 8%/0.1% | 1000 ul | 250 ul | 50 ul | 3700 ul | ?? kN/m2 |
| 8/0.08 | 1000 | 200 | 50 | 3750 | 75 |
| 8/0.06 | 1000 | 150 | 50 | 3800 | 30 |
| 8/0.05 | 1000 | 125 | 50 | 3825 | 23 |
| 8/0.04 | 1000 | 100 | 50 | 3850 | 17 |
| 8/0.03 | 1000 | 75 | 50 | 3875 | 14 |
| 8/0.02 | 1000 | 50 | 50 | 3900 | 10 |
| 5/0.12 | 625 | 300 | 50 | 4025 | 33 |
| 5/0.10 | 625 | 250 | 50 | 4075 | 28 |
| 5/0.08 | 625 | 200 | 50 | 4125 | 24 |
| 5/0.06 | 625 | 150 | 50 | 4175 | 15 |
| 5/0.05 | 625 | 125 | 50 | 4200 | ?? |
| 5/0.025 | 625 | 63 | 50 | 4262 | 7 |
| 3/0.10 | 375 | 250 | 50 | 4325 | ?? |
4. Add 200 ul of the FITC dextran stock and 10 ul of acrylic acid. Degas 30 minutes.
5. Add 30 ul ammonium persulfate, 20 ul TEMED. Mix gently by gentle swirling.
6. Immediately add 100 ul of the acrylamide mixture per ml of AOT while stirring. Allow acrylamide to polymerize for 1 hour while gently stirring (setting 2) under a gentle stream of nitrogen, which should barely move the surface of the liquid.
7. Spin in the Sorvall at 500 rpm for 5 minutes and remove the supernatant. Add 8-10 ml MeOH. The beads should remain insoluble. Repeat the spin and resuspension procedure with MeOH 3-5 times, then with PBS for 3-5 times and resuspend the final pellet in PBS. The beads may be stored at 4oC indefinitely.
8. Take 100 ul of stock bead solution, add 400 ul of MES buffer. Spin at 2,000 rpm for 2 minutes in an Eppendorf microfuge, collect the supernatent in a fresh tube and spin at 14,000 rpm for 2 minutes to collect the beads that are 1-10 microns in diameter.
9. Spin and resuspend the beads 3 times with 1 ml MES buffer for each wash in the Eppendorf microfuge as above.
10. Add 0.5 ml of 26 mg/ml EDC in MES, shake on a rocker for 2 hours.
11. Spin and resuspend the beads 3 times with MES buffer as in step 9.
12. Add 1 ml of the protein for coating and mix on a rocker overnight.
13. Spin and resuspend the beads 3 times with 1 ml PBS for each wash. Beads may be stored at 4oC for up to 2 weeks, depending on the longevity of the coated proteins.
[Home] [People] [Projects] [Resources] [Protocols] [Publications] [Video] [Contact]