I am having trouble purifying my S-tagged protein. All protocols I have read call for batch mode, but say that column mode is also doable. I am using a column with S-protein Agarose from Novagen, loading it with my supernatant, and eluting with magnesium chloride. I do not see anything in the elution fractions in SDS-PAGE gels, not even when I silver stain. I performed a WB using an antibody specific to my protein and it shows the protein in the flow through fraction. I think my protein is not binding to the column but I am not sure. Can anyone provide insight? Has anyone been successful at this?
Hi Arianna.
A few years ago I did some work with S-tagged proteins and had the same problem you are facing now. I was able to overproduce the protein and it was highly soluble but its purification was... traumatic to say the least.
Novagen's protocol recommended the purification to be performed in batch mode. I did that initially but was losing large amounts of protein and the elution fractions had a large number of different sized proteins that were being purified together with my protein. Adding to that, this batch mode was making me lose a large quantity of the S-protein agarose (after two purifications I had about half of my initial resin volume).
Thus, I decided to incubate the protein extract with the S-protein agarose for 30 minutes and pack the resin with the bound protein in a column. Then I washed the unbound proteins and performed the elution with 3M magnesium chloride.
This time I was satisfied with the results. Not the best protein purification ever but I was able to obtain a good amount of protein and there was only a very small trace of other proteins.
The problem was that when I reused this packed resin for a new purification I obtained absolutely no protein!
So my suggestion is that whenever you need to purify an S-tagged protein you must incubate your supernatant with the S-protein agarose and then pack it in the column before eluting. Then you can try to unpack the resin and use it again.
I hope this is helpful. Good luck.
there are various factors that can play during purification. If the protein is expressed well (in soluble fraction), and not binding to the partner beads, then I would prefer to look for the buffer used for binding. You may try adding 0.2% triton X-100 in your lysis buffer, this may keep your protein happy and tags to be exposed for binding.
More info on your current buffer system would be helpful!
good luck.
Try to fractionate your supernatant with ammonium sulfate first. http://www.ncbi.nlm.nih.gov/pubmed?db=pubmed&cmd=search&term=14725509
Hi Arianna.
A few years ago I did some work with S-tagged proteins and had the same problem you are facing now. I was able to overproduce the protein and it was highly soluble but its purification was... traumatic to say the least.
Novagen's protocol recommended the purification to be performed in batch mode. I did that initially but was losing large amounts of protein and the elution fractions had a large number of different sized proteins that were being purified together with my protein. Adding to that, this batch mode was making me lose a large quantity of the S-protein agarose (after two purifications I had about half of my initial resin volume).
Thus, I decided to incubate the protein extract with the S-protein agarose for 30 minutes and pack the resin with the bound protein in a column. Then I washed the unbound proteins and performed the elution with 3M magnesium chloride.
This time I was satisfied with the results. Not the best protein purification ever but I was able to obtain a good amount of protein and there was only a very small trace of other proteins.
The problem was that when I reused this packed resin for a new purification I obtained absolutely no protein!
So my suggestion is that whenever you need to purify an S-tagged protein you must incubate your supernatant with the S-protein agarose and then pack it in the column before eluting. Then you can try to unpack the resin and use it again.
I hope this is helpful. Good luck.
Traumatic purifications.....I know the feeling!
Thank you so much! I will try this!
S-tags are not that useful for large-scale (i.e. mg) I would say. Ok for small-scale, but move to His-tag or non-tagged purifications for large-scale workups.
- Badges
- Science method