I am trying to separate some bee venoms by HPLC. After runs, there is residual protein on the column, even after multiple washes from 5 to 100% ACN. Although there is no mention of this in recent (last 20 years) papers, older papers say that melittin (a major protein of many bee venoms) aggregates and sticks to columns. However, there is no mention of how to get it out. In fact, recent papers don't even discuss the issue at all, as if they are not having the problem. Does anyone have experience with this? Thanks!
Ryan: If its an accumulation of aggregates, one simple way is to reverse the connections and repeat with your process. Good luck.
If it is stuck in your reverse phase column may be the hydrophobic part of melitin which is responsible. It is better to used organic solvent like hexane.
Hey Ryan - we use an injection of 100% isopropyl (50-500 uL) on a regular basis to clean off retained materials. Run at 100% B and then do an injection or two - seems to help.
Thanks for all the suggestions, everyone. I have run the column up to 100% MeOH followed by 100% isopropanol and 100% hexane. Then I have come back down in reverse order. This has been done twice, but the column still has residual protein coming off. This is per the manufacturer's instructions. The amount coming off has gone down, but is still detectable and doesn't seem to want to go any lower. Reverse flow is not advised for one of our sticky columns, but has also been done on another, again with protein still stuck.
Are you including acid in the mobile phases? If not try using 0.1%v/v Trifluoroacetic acid or 0.1%v/v Formic acid and running a gradient from aqueous to organic (Acetonitrile) with the acid in both mobile phases.
Hi,
If all organic washing suggestions provided above don't work, you could consider incubating your column with a protease, such as pepsin, to cleave the protein/peptide that is clogging your column; the resulting peptide fragments are likely to be eluted by the gradient you'd routinely use for your analyses.
Good luck!
As Bruce suggested, you may add some acid to your mobile phase. 0.1 % TFA or HCOOH is commonly used. Be careful with basic conditions, this may destroy your column material. Some organic buffers may help (e.g. HEPES) adjusted to pH 7-8, this increases the solubility of the protein but does not destroy the stationary phase. At first, wash with 10 column volumes 100% H2O, then add 5-10 column volumes buffer at low flow rate (0.05-0.1 ml/min), remove buffer with H2O + 0.1 % acid (20 column volumes), then try the organic phase again (with acid added).
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