Size exclusion chromatography for resolution of 18 kd protein and 15 and 10 kd d - columns for the range? (Aug/04/2008 )

hii..
i need to develop a size exclusion chromatography for resolution of 18 kd intact protein and 15 and 10 kd degraded products of the protein.
any suggestion for columns for the range for which i can expect resolution?
thanks!!

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Hi,
I'm using a TosoHaas TSKgel G2000SW 7.5x60.0 cm on a HPLC system, for separation of peptides of 20, 15 and 5 kDa.

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GE used to make a peptide column, exclusion size 20 kDa. Really nice resolution.

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Why use size exclusion chromatography? This is highly unlikely to work as the MW of the proteins are too similar. You would be better off using a technique that has higher resolution such as ion exchange or hydrophobic interaction chromatography.
If you have problems with degradation you could also try the following steps earlier in the protein prep.

1. Add 100 micromolar AEBSF (a serine protease inhibitor) to the cell suspension before you lyse the cells. You can store aliquots of this in the freezer for up to 6 months.
2. Sterile filter all buffers used for the preps into autoclaved glassware and after each purification step sterile filter the protein solution to ensure that no bacterial/fungal or other contamination is present. Sterile filtering won't remove contaminating proteases though.
3. Avoid contaminating your protein prep yourself. Don't touch any tubes containing protein without wearing gloves as your hands are covered with bacteria. It never ceases to amaze me how many people don't wear gloves when they put fraction tubes in the fraction collector.
4. Add 1mM EDTA to your protein solution (obviously you can't do this is your protein has a metal ion in it that is required for its function and you can't have EDTA in the buffer if you are using metal affinity chromatography (His-tag purification) as your initial purification step but you can add it to buffers used later in the prep or to your storage buffer. EDTA will inhibit metalloproteases.
5. Do the prep at 4oC to reduce proteolysis.

I would try ion exchange to see if that can clean up the protein (see the GE Life sciences ion exchange handbook for how to optimise the pH of the buffer for this technique). Many proteins precipitate in low salt buffers required to get it to stick to the ion exchange resin so test this on a small scale first. For example use prepare 2 mL solutions of your protein at 0.1 mg mL-1 and load these onto 1mL Q-sepharose or 1mL SP sepharose (depending on the whether the calc pI of your protein is <7 or >7 respectively) at various pH conditions between 4 and 9.
Find the pH where your protein just sticks to the resin.

If ion exchange doesn't work then try Hydrophobic Interaction Chromatography (HIC). GE life sciences sell a nice HIC selection kit which has a selection of 1mL HIC columns that you could test to find the one that works best for this protein.

Otherwise clone the protein into a different vector that contains multiple affinity tags. I regularly use vectors available from EMBL (Hamburg) which have both a His-tag and a GST tag that can then both be removed by TEV protease cleavage (a highly specific protease). Using this system you can use Ni resin and immediately follow this with a GST resin purification (or the other way around) so that you can very quickly get highly pure protein that should be protease free (within a few hours after lysing the cells). Cleave off the tags using a His-tagged TEV protease overnight at 4oC and then pass the protein solution through a Ni resin column. The TEV and the cut His-GST tags bind to the resin whilst your protein of interest passes straight through. Very easy and compatible with most buffer systems.

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