This column was previously packed, what I'm estimating to be 2-3 years ago. There appears to be a crack of air in the column, but other than that it looks relatively even. When I run my sample on it, which is a protein and it's degradation products of >15 kDa difference in size, they aren't separated even in reducing conditions. There also doesn't appear to be a void volume, but I see multiple small peaks other than my protein, so I'm confused. Should I go about repacking it? Also, does anyone know what other supplies I'd need to repack it? Would a deep wash be sufficient?
Dear Ryan,
If there is any gap, crack or bubble in your column you should definitely repack it. Provided there has been no microbial growth (in which case you should dump its content and pack your column with fresh resin), recover the resin, resuspend it in water, degas and repack you column according to the manufacturer's instructions. Also, even with a well packed column, don't be too optimistic about the resolution : gel filtration columns don't afford a satisfactory separation unless the size difference between the species to be separated is at least 40-50 % (e.g. BSA and ovalbumin)
When there are known problems or doubts, just replace the resin with fresh material or clean, regenerate and re-pack with the previous material. In either case, please be sure to run test stds through the column to make sure it is acceptable BEFORE you start to use it with real samples (i.e. construct a calibration table).
*For the data to have any chance at being valid, you always need to start with known good materials and appropriate methods.
What you really want to do is SE-HPLC with MALS-UV as detectors: even if you will not be able to completly resolve the peaks of full length protein and its degradation product, you will be able to determine changes in molecular weight in separate slices of your SE peak. Furthermore, MW determined by MALS is accurate (if properly determined, which is not trivial), while the value obtained by gel filtration is only an approximate indication.
If you need to separate the two species, ion exchange is your best option, or affinity purification using mAbs in the case you have domains specific antibodies.
P.S. If you have a crack in a GF column definitely repack it and measure resolution and theoretical plates of the column. It's easier than what you might think.
- Badges
- Science method