Hi all, I need some help.
I am trying to purify and characterize an extracellular killer toxin (protein) from the yeast W. anomalus. But I am having trouble with SDS-PAGE analysis of the purified protein. The protocol was as follows:
I concentrate the cell-free supernantant 100x by ultrafilitration (10 kDa) in presence of 4M urea to avoid aggregation. Then I purify the protein by SEC in a G-75 column with 50 mM citric acid-phosphate + Urea 4 M. The protein elutes in the dead volume as a single peak of absorbance at 280nm. It is detected by its antimicrobial activity. So far so good, but when I run the sample in an 12% SDS-PAGE gel, it shows smears and no discrete band is observed.
I´ve also tried adding 8M urea (final concentration) to the purified sample before boiling it (or not) in laemmli sample buffer, but no differences were observed.
Is it precipitation?? could the smears be a running profile of the same protein with slight differences in glycosylation?
I`d appreciate any suggestions.
It is more likely that you are running out of sds. Sds must always be in excess over the protein. Increase sds concentration or decrease the amount of protein. Also do not boil the samples.
The problem may be that the medium contained high molecular weight material, such as carbohydrates or nucleic acids, released by the bacteria, and your protocol has concentrated them and not removed them, since your protein eluted from the SEC column in the dead volume, where anything large would also elute (which suggests that your protein is aggregated despite the urea, by the way).
You could try treating your sample with nucleases to destroy any nucleic acids. You should also add a chromatography step that involves binding the protein, such as ion exchange chromatography, to separate it from the contaminants.
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