The PD-10 columns are made of chemically inert polypropylene material and filled with 8.5 mL of Sephadex-25 gel filtration material. The exclusion limit of the Sephadex-25 is 5000 for globular protein. The column can be fitted with the LabMate buffer reservoir, which is very useful when equilibrating the column with buffers. The LabMate reservoir allows one to add up to 25 mL of buffer at a time; thus, eliminating the need to add buffer to the columns in small aliquots. Instead, one can add the buffer and take care of some other work in the mean time.
There is no need to pretreat/prewash the columns; just attach the LabMate reservoir to the top of the column and add 25 mL of the desired buffer to equilibrate the column. Once the column has equilibrated, add 2.5 mL of the protein sample to be desalted/buffer exchanged. If the sample volume is less than 2.5 mL, make up to 2.5 mL with the buffer. For sample volumes greater than 2.5 mL, aliquots of 2.5 mL can be processed either batchwise (reusing the same column after washing) or by using more than one column. Discard the flow-through. Then add 3.5 mL of buffer at a time, and collect 2 mL fractions, 5 fractions are usually sufficient. Most of the protein will be in fractions 2 and 3. Finally, wash the column with 25 mL of distilled water. Now the column is ready for reuse.
We have used a single column at least 5-6 times to desalt a GFP-fusion proteins (5 mg/mL) without any loss of efficiency in desalting or loss of protein, with reproducible results. Our GFP-fusion proteins were in His-Trap elution buffer, containing approximately 250 mM imidazole and 500 mM NaCl in 50 mM sodium phosphate buffer, pH 7.4, apart form 1% CHAPSO. During desalting on the PD-10 columns, proteins were diluted approximately 2.5 fold. GFP-fusion proteins were then concentrated using a Vivaspin (Sartorius) centrifugal concentrator. When further purification of the protein was necessary, the desalted fusion protein from the PD-10 column was purified by passing through Mono Q column (also from GE Healthcare).
The PD-10 columns are a simple and efficient tool to desalt protein samples. Since the same column can be used repeatedly over and over, they are very economical too.
| Comment on this Review |
Comments? Questions? Discuss this review of Disposable PD-10 Desalting Columns From GE Healthcare with the author of this review, Dr. Jaganathan Lakshmanan, and the rest of the Biocompare community.
on Feb 27, 2008 salman butt said:
But if you run it through a monoQ, won't you need to desalt it again due to the elution conditions requiring NaCl? Or is there another way of doing so?
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Dr. Jaganathan Lakshmanan
Research Associate
Neurobiology Lab, Department of Psychiatry
University of Louisville
United States
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