Sepharose-4B covalently attached with monoclonal antibody of certain protein tags are used to isolate fusion protein with such tags.

Gel preparation

Protein binding

Protein Elution

Detection

Thoroughly suspend the affinity gel in the vial, in order to make a uniform suspension of the resin. Quickly transfer 10 μL of the gel suspension (about 5 μL of packed gel volume) to a fresh tube. Add 0.6 mL TBS and thoroughly suspend the gel. Centrifuge the resin at 10000 rpm for 30 s. Remove the supernatant carefully and make sure most of the wash buffer is removed and no resin is discarded. Repeat for 3-4 times.

Add protein samples to the washed resin. Agitate all samples gently for 2 h at 4℃. Centrifuge the resin at 10000 rpm for 30 s, and remove the supernatants to a fresh tube. Wash the resin with 500 μL TBS until the OD280 of the supernatant liquid <0.05.

Add proper volume of affinity elution buffer or 0.1 M Gly-HCl to the resin. Agitate gently at proper temperature for proper time. Centrifuge the resin at 10000 rpm for 30 s, and remove the supernatants to a fresh tube (When using Gly-HCl, 1/10 volume of 0.5 M Tris-HCl with 1.5 M NaCl should be pre-added in the tube.). The supernatants can be kept at 4℃ for immediate use or -20℃ for long term preservation.

Add 0.1 M Gly-HCl to 3 times volume of the resin immediately after use if regeneration is needed. Agitate gently for 3-5 min. Centrifuge the resin at 10000 rpm for 30 s. Remove the supernatants and add TBS to 3 times volume of the resin. Agitate gently for 2-5 min. Centrifuge the resin at 10000 rpm for 30 s. Collect the supernatants and measure the pH value. When pH value reaches neutral, add TBS with 50% glycerol to 10 times volume of the resin to wash, and leave no less than the same volume of the buffer, preserve at 4℃ or -20℃. Repeat to add TBS and centrifuge when the pH value is still acidic.

Add Laemmli 2X buffer to the same volume of both the resin and the supernatant and boil the samples for 5 min. Load samples on SDS-PAGE and detect the tagged protein using respective monoclonal antibody.