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Protein Dialysis, Desalting, and Concentration

Protein dialysis, desalting, and concentration are crucial steps in order to well prepare your protein samples before running downstream proteomic applications such as mass spectrometry, NMR spectroscopy SDS-PAGE, x-ray crystallography and isoelectric focusing, etc. Lysates always have contaminants that are incompatible with downstream assays and applications. Creative Biostructure handles various resins and devices to desalt and remove detergents from samples simply and efficiently.

Dialysis is one of the most frequently used separation techniques to facilitate the removal of small, undesired materials from macromolecules in solution by a selective semi-permeable membrane. The size of pores of these semi-permeable membranes determine their molecular-weight cutoff. The principle of dialysis is that sample molecules will be retained on the sample side since they are larger than the membrane pores, while small molecules diffuse freely to reach an equilibrium concentration. As a result, the concentration of small unwanted molecules decreases to negligible levels, while the concentration of the sample increases to desired levels.

A Typical Dialysis Procedure

  1. Prepare the membrane under instructions;
  2. Sample loading by using dialysis tubing or device;
  3. Dialyze for about 1-2 hours (room temperature);
  4. Change the dialysis buffer, then dialyze for another 1-2 hours;
  5. Change the dialysis buffer again, then dialyze overnight (4°C).

A Typical Protein Desalting Procedure (for Zeba Desalting Columns)

  1. Remove the column’s bottom closure;
  2. Put spin column in a 1.5 mL collection tube;
  3. Centrifugation (1500 g, 1 minute) to remove the storage solution;
  4. Put a mark on the side of the column, and keep the mark facing outward all the time for centrifugation;
  5. Add 300 μL of 1X Modification buffer (pH 8.0) for a centrifugation (1500 g, 1 minute), discard flow-through;
  6. Repeat step 4 and 5 for another two rounds, and discard buffer in each round;
  7. Protein sample loading (up to a 130 μL sample volume);
  8. Centrifugation (1500 g, 2 minute), then collect desalted sample;
  9. Discard the used column.

Troubleshooting:

1. Protein samples are too dilute for further application.
You may try the following ways to concentrate your protein samples:

  1. a. Centrifugation
  2. b. Lyophilization
  3. c. Precipitation
  4. d. Dialysis

2. Significant protein sample lost.
Please make sure that your targeting protein sample is at least 2-fold larger than the MWCO to achieve a high protein recovery. For example, a 5 K MWCO device would be appropriate if your protein sample is larger than 10 kDa.

3. Leftover salts in the protein sample.

  1. a. Reduce the volume of protein sample processed in your column;
  2. b. Run the flow-through in a fresh column for another desalting round.

4. Filled flask does not float.
Please make sure that the flask capacity was proper and the flotation ring was properly attached.

5. Contaminants removal is not completely.
Molecules differ in diffusion rates across membranes, some molecules do not act as other molecules of similar molecular weight. To enhance the concentration, you can try the following steps:

  1. a. Increase dialysis time;
  2. b. RPerform with several buffer exchanges;
  3. c. Use a device containing a higher MWCO membrane.

Besides Protein Dialysis, Desalting, and Concentration, Creative Biostructure is also able to help your protein purification project with technical resources and supports. We are pleased to accelerate your research. Please feel free to Contact Us.


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