I've recently run chromatography on Strep-Tactin column, but the activity was only in flow through fractions (75% total; no activity in elution).
The sample was in 200 mM Tris/HCl pH 8.0 with protease inhibitors (it was old about 3 or 4 weeks).
Buffer A: 100 mM Tris/HCl, pH 8.0; 150 mM NaCl (as recommended)
Buffer B: A + 2.5 mM desthiobiotin
Flow rate 3 ml/min (with 5mL column)
Basically I followed the recommended procedure, but both Western blot and activity measurement confirmed that the protein is present in first two fractions and nothing in the elution, so obviously it didn't bind at all.
All I could find in troubleshooting was either usage of protease deficient E.coli strain or protease inhibitors to limit degradation or increasing concentration and amount of the protein to prevent some leaching or something. However, I don't think that would be problem, since I had all the activity in flow through, it wasn't lost.
My only idea is to decrease the flow rate during loading of the sample. However, is there anything else we could try to enhance binding to Strep-Tactin column?
Yeah, I think so (it's not my construct, it's work for somebody else, so I'm not tat much familiar with all the details and I'm quite confused about all the versions of Strep-tags).
Did you do the western with a Strep-tag antibody? The Strep tag can often be digested, but it is unlikely, when you used a Strep-tag antibody!
If you did use a protein specific antibody, digestion might be a problem. To avoid this you can use a double Strep II tag (SA-WSHPQFEK(GGGS)2GGSAWSHPQFEK).
Also, did you check if the column is still good (by seeing the color change when applying HABA buffer fro yellow to red)?
I used anti HA antibody. However, the order is POI-HA-Strep-tag, so theoretically it could be cleaved off. However, how to find out that? I don't think we have Strep-tag antibody.
The column was brand new.
Well I don't have that much experience with Strep tags but I have purified a lot of proteins with His tags and the problem in cases like these is that the tag is not accessible. Have you tried putting the tag at the N-terminus?
So I have confirmed the presence of the Strep-tag on the protein by antibody yesterday.
The C-terminus should be on the surface (actually both termini close to each other), so it should be OK. It was not given to N-terminus because of possible targeting peptide.
Theoretically yes, since I was trying to determine oligomerization status of other homologue previously. But that one has quite different properties (transmembrane) and no other such data were published so far.
Dear Andrei
For all people interested in Strep Tag, could you remind us the difference between Strep Tag, Strep Tag II and Twin Strep Tag ? Just for our records. As IBA only mentions Strep Tag II, I can not find sequence information for the "ordinary" Strep Tag.
Thanks
Beate
Check whether the column is active with HABA. If the column becomes red,it is OK. then there must be some with the strep-tag or your protein
I did many IPs using Strep Tactin and had the same problem when the sepharose was stored not properly or has gotten too old. I guess you have to check that column is still working. Flow rate would not matter as I always do gravity flow binding which has a considerable higher flow rate compared to your settings. If you have some Strep-Tactin Sepharose you can also pour it over, quick wash and cook the beads to see whether you have a general binding problem, which may arise from folding/aggregation and shielding the tag. This would give you a quicker result that optimizing a whole LC pipeline and try to find the problem.
Well, the column is somewhat orange-ish, I wouldn't probably call it exactly red. But hey, I'm just a guy ;)
@Timo Glatter: The column was bought brand new. And to check that it works I tried it also with other proteins which were previously purified via this means. However unsuccesfully.
I'm sorry, I didn't understand you last suggestion.
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