Molecular Biology. Practical Guide - Velikov V.A. 2013

Isolation of recombinant protein
Dialysis of the protein preparation

The periplasmic protein isolated via the borate method contains significant amounts of low-molecular-weight impurities, primarily salts. These can be removed by dialyzing the preparation. The protein solution is placed in a dialysis tubing whose walls act as a semipermeable Cellulose membrane that retains molecules larger than 15-20 kDa. The bag is then immersed in a buffer solution. The volume of the external buffer must significantly exceed the volume inside the tubing. Due to osmosis, low-molecular-weight substances pass through the membrane into the buffer. The desalted protein solution, containing molecules larger than 20 kDa, remains inside the dialysis bag.

Materials and Equipment

Protein preparation, dialysis tubing, 2000 ml beaker, magnetic stirrer, Glass rod or pipette.

Solutions

- PBS buffer. For the preparation of 1 L: NaCl - 5.84 g; Na2HPО4 - 4.72 g; NaH2PО4 х Н2О - 2.64 g; pH 7.2.

Procedure

1. Transfer the protein solution into the dialysis tubing using a micropipette.

2. Tie off the tubing and secure it to the middle of a glass rod or pipette using a short piece of rubber tubing or a rubber band.

3. Submerge the bag into a 1000 ml beaker filled to the brim with PBS buffer, resting the glass rod across the rim of the beaker. Place the beaker on a magnetic stirrer located in a refrigerator and turn it on.

4. The following morning, replace the buffer with fresh buffer and continue dialysis for another 4–6 hours.

5. Determine the protein concentration. The resulting scFv minibodies are suitable for dot blotting, Western blotting, and immunodiffusion.



Last update: 13/08/2026

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