Polyacrylamide Derivitization Overview
This protocol describes how to successfully coat the channels in a chip with a covalently-attached layer of polyacrylamide. The procedure occurs in 3 steps: cleaning with NaOH, silanizing with “gamma”, and completing the coating with polymerizing acrylamide.
Reagents Needed for Polyacrylamide
• 1 M NaOH • gamma-methacryloxypropyltrimethoxy silane (“gamma”) • Methanol • Acetic Acid, pH 3.0-3.5 (~0.1% v/v) • Acrylamide (electrophoresis grade) • ddH2O – MilliQ • 10% Ammonium Persulfate (APS, made fresh or kept frozen) • TEMED • Argon
Polyacrylamide Derivitization Procedure
1. NaOH cleaning: Using a syringe, fill the channel to be derivatized with 1M NaOH, flushing through ~ 100 uL. Leave for 1 hour. (Further flushing is optional)
2. Sparging acrylamide: Make a 5 mL solution of 3% w/v acrylamide in ddH2O in a 20 mL scintillation vial. Cap it with a rubber septum, and insert two syringe needles through the septum. One should be long enough to reach into the fluid for bubbling argon, the other serves as a vent for the gas. Flow argon through the larger syringe needle so that it bubbles through the acrylamide solution. Sparging should proceed for at least 1 hour.
3. Essential Flush: Flush the channel thoroughly with the acetic acid solution – at least 200 uL.
4. Silanization: Prepare a 0.6% v/v solution of gamma in 30% methanol as follows. In an eppendorf tube, add 6 uL gamma to 300 uL methanol. Mix briefly, and add 700 uL acetic acid soln. (The order is important, because gamma is not soluble in water, but is in methanol) Immediately flow this solution into the channel, flushing through ~ 100 uL. Leave for 1 hour.
5. Acrylamide Binding: To the sparged acrylamide, add 25 uL 10% APS (final concentration 0.005% w/v or 5 uL/mL), and 5 uL TEMED (final concentration 0.1% or 1 uL/mL). Mix briefly and gently, and immediately flow the solution into the channel, flushing through ~ 100 uL. Leave for 5-30 min, then thoroughly flush channel with ddH2O or the buffer of your choice.
Notes from Stephanie Yeung
- The Hjerten coating should be good for many, many runs (10-50), but generally not good for more than a month
- Cap the polymerizing acrylamide solution after you use it. Check it later to see if it has noticably polymerized; the viscosity will increase if polymerization occurs. Even a very minor change in viscosity is indicative of polymerization.
- Remember to dispose of the polymerized LPA in the non-halogenated solvent containers in the hood.
- Use caution when opening TEMED near other people. It smells very bad.
- Eric Chu 21:25, 22 July 2009 (PDT):
or instead, discuss this protocol.