I work in a lab with access to the NGC FPLC from biorad and the duo-flow. I want to set up a protocol for size exclusion and desalting. I was wondering if anyone has helpful tips for the set up. I want to buy the resin and reuse it for separating proteins in the range of 11-130 kDa. I have the MT-5 and MT-10 columns from Biorad as well.
What resin should I get that would be compatible with columns that have a 5-10 cm length.
Hi, I think, that this link could be useful for you. This free e-book is quite comprehensive manual of SEC. It was very helpful for me. Enjoy :).
http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc18102218_20141208003707.pdf
Hi there,
For this range of resolution, I work with Superdex200 from GE (16/60) preparative column at 1mL/min. Bed volume is 120mL so experiment is 2h long. For optimal resolution sample should be less than 5% bed volume and the column equilibrated with at least one bed volume. The size of column you are mentioning is not compatible with efficient SEC unless the column has very thin section (for instance, the column I use for analytical purpose is still 10/30... However it sounds OK for affinity chromatography, hydrophobic chromatography or IEC. Or just desalting (like what can be done with PD-10 columns).
Hi, I think, that this link could be useful for you. This free e-book is quite comprehensive manual of SEC. It was very helpful for me. Enjoy :).
http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc18102218_20141208003707.pdf
For separation of proteins from each other (high resolution gel filtration) and for separation of proteins from salts (group separation) you need different columns. Different media in the column and different length. Read the book Jiri linked to above and you will have all your answers.
P.S. FPLC is a brand of GE, so what you have from BioRad is not an FPLC :)
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