Chromatography Forum

Hi
I am not sure if this is the right place to put this question, please let me know if there is any more appropriate place...
I am assaying a recombinant protein for its enzymatic activity and wish to extract the products (hydrophobic) through reverse phase solid phase extraction cartridge. For doing this, can I directly put my assay reaction (having the protein ) onto the SPE column? Is it necessary to remove the protein before purification? Can the protein interfere with the purification of product? In case of hydrophilic products of recombinant enzyme, we deproteinate the assays by washing with chloroform (not for SPE purification), but in this case, since the products are hydrophobic, they too will go away with chloroform Later, I would be analyzing the products on GC
Many thanks!
Ram

I believe that you can get "internal surface reversed-phase" materials in SPE format. These are essentially silica gel which has reversed-phase (hydrophobic) functionality only on the inside of the pores, with the outside of the particles being hydrophilic. The idea is that the proteins are large enough to be excluded from the pores, while small molecules can enter and be retained. I know Regis Technologies sells (or at least sold) that material in HPLC format; they might be a good place to start.

I use ZipTips to 'rid' my reactions of the enzyme. It's easy and quick. It could work well for you..as it seems that you know what the products of the enzymatic reaction are.

But Tom's product advice is quite interesting. I've never heard of that product before.... I might try it sometime!!!

To get some ideas you can look at papers by Karl Siegfried Boos on the use of restricted access materials RAM, which is the technique described by Tom.

Merck offers LiChrospher® ADS columns that allows fully automated, LC integrated sample preparation of hydrophobic, low molecular weight analytes from untreated samples like serum, milk, fermentation broth of food homogenates. The enrichment is based on two chromatographic processes: reversed phase and size exclusion chromatography since LiChrospher® ADS sorbents are spherical silica gel particles (25µm) with DIOL modification on the exterior surface and alkyl modifications on the surface of the interior pores. Depending on the hydrophobicity of the analyte three types of precolumns are available: C-4, C-8 and C-18.

More information can be found at http://www.merck-chemicals.com