Protocol for Making PAAM gels with NHS ester
Padma Rajagopalan
1. Add the desired volume of 40% acrylamide solution ( I add 2 ml)
2. Add the desired volume of 2% bis-acrylamide solution ( I add 0.2 ml)
3. Add 15 microliter of TEMED
4. Add 2 ml of 0.25M HEPES
5. Mix briefly using the vortex mixer
6. Check pH of solution (it will be around pH 7)
7. Add 1N HCl (drop by drop) until pH is 6 (if it is a little below or above 6 it is ok e.g.
pH range 5.9 to 6.1)
8. Add DI H2O until total volume is 10 ml. Vortex the solution.
9. Add beads if doing traction experiments. Usually 250 microliter beads for 10 ml
solution. Sonicate beads for 10 minutes before adding to PAAM solution to minimize
bead agglomeration in the final gel.
10. Vortex the solution after adding beads.
11. Add the NHS-ester, I usually add 10 micromole/ml of reactant solution.
(the ester is toxic , wear gloves when handling this chemical!!)
Once you are done measuring the ester, put the bottle back in the refrigerator
immediately.
12. Vortex the solution.
13. Add 50 microliter of Ammonium Persulfate solution (0.1g/ml)
14. Vortex the solution
15. Pipet 10 microliter (if using modified 12-well plates) or15 microliter (if using 25mm
coverslips). Place a 12 mm coverslip on the top of each gel (if using 12-well plates) or a
18mm coverslip (if using 25mm coverslips as the substrate).
16. Wait 10-15 minutes. Remove the coverslips.
17. Add the protein/peptide solution. Add the protein/peptide within 20 minutes of
making the gel.
18. Store in the refrigerator. If adding protein/peptide, you must incubate for 18-20 hours
in the refrigerator.
19. Remove from the refrigerator, rinse once with DI H2O.
20. Add 50 microliter of ethanolamine solution (50mM in HEPES @pH8). Wait for 1
hour.
21. After 1 hr rinse the gels for an additional hour in cold DI H2O.
22. UV Sterilize gels for 30 minutes and plate cells as per standard protocol.