Activated CH4B Sepharose (Pharmacia) 5/10/01Beth

Pre-activated gel for covalent immobilization of proteins and other ligands containing primary amino groups.

Prior to coupling: Make sure your protein is not in Tris, a good coupling buffer made fresh (if older the buffer gives off CO2 and gets more alkaline) is: 0.1M NaHCO3, pH 8.0 containing 0.5M NaCl. You can use PD10 columns or dialysis to change the buffer. Basically most buffers I have used at pH 8.0 work well. You may have to add 0.5M NaCl to improve coupling but try it without first. Take a gel sample of pre-loaded protein. Save the flow-through to re-couple if all doesn’t couple in the first trial, take a gel sample of this too to monitor coupling or use OD.

1) Weigh out proper amount of freeze dried gel (always parafilm bottle after use to keep it dry). 1g swells to around 3 mls of resin.

2) Have 1mM HCL pH 2.0 cold and rready to use. Wash with around 200mLs HCl per 1 gram of swelled gel. To WASH: Start a timer. Add a few mLs of HCl to the weigh boat containing the resin, then either transfer to a glass sintered filter or if using a large volume add to a beaker containing more HCl, swirl and pass over a larger glass sintered filter. Make sure that the flow rate of the vacuum is not too hard that it dries the resin out! Do not wash longer than 10 minutes (in the brochure they say 15 but I have done this and coupling efficiency decreases tremendously with every minute longer. Once you have washed the appropriate volume of HCl through the resin, collect the resin in the bottom of the filter and turn the vacuum off. Lift the top part of the filter off and scoop directly into your protein that is in a screw top falcon tube. (The brochure wants you to wash the resin first with H2O before adding it to your protein but again, the efficiency goes down so don’t do that). As long as your protein is in a good buffer the pH will not go down but check to be sure when you do this.

3) Rock gently at 4°C O/N

4) The next day collect AND SAVE the flow-through by either spinning the beads softly (they break at too high speed) or let the beads settle in the solution on ice while you get the blocking buffers ready.

5) To block: alternate washes of the beads in a glass sintered filter with these two buffers. Repeat 3X. This will get rid of unbound protein and block any left-over active groups on the beads. This step is absolutely necessary.

0.1M Tris, 0.5M NaCl, pH 8.0 alternate with: 0.1M NaAcetate, 0.5M NaCL, pH 4.0

For a final wash keep the protein in the Tris buffer or put it into your favorite buffer.

NOTES: Using a slightly lower pH is better in some situations since hydrolysis of the active ester is minimized. Alpha-amino groups are preferentially coupled at lower pH as opposed to epsilon amino groups due to a lower degree of protonation of alpha amino groups at lower pH.

Optimal coupling times: 1-2hrs RT

4hrs 4°C (I do O/N for convenience).

Active group: N-hydroxysuccinimide with a 6-aminoheanoic (6 atom) spacer.