I have fractions collected of my protein from FPLC (liquid chromatography) and I am wondering how I am best able to load my samples on a SDS-PAGE gel after lyophilizing/freeze drying the fractions? Any one have a protocol for this?
Compared to HPLC, the elutions obtained from the FPLC are loaded with salts, after lyophilizing and resuspending those samples in a smaller volume, these salts concentrate and interfere with the sds Page (the bands will look wavy). So you would need to desalt them after restorage by dialysis, or precipitating a sample with TCA/acetone
Dissolve lyophilized protein pellets using Leammli buffer directly if the conc is low and silver staining is a good option to increase protein band visibility. If still, the conc is low to monitor in SDS-PAGE then several injections and collecting the multiple fractions, pooling followed by lyophilization may be an option. Starting with concentrate sample and large volume higher scale purification columns can also be considered. Ultrafiltration is also good at both concentration and for desalting, if you perform wash replications to proteins on the membrane.
Good luck,
Emir...
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