I express my protein in pTYB12 vector as fusion with intein and then try to purify on chitin beads.
Typically, I load overnight at low flowrates (something like 0.3 ml/min). The loading buffer is 50 mM Tris/HCl, pH 8.0, 1 M NaCl, 1 mM EDTA, 5% glycerol, 0.1% Triton X-100. Afterwards, I wash with buffer without Triton. Of course I work at 4°C.
Anyway, typically, I have something like 30% of activity in unbound fraction and 30% in purified fraction; the rest is lost somewhere.
I don't think the problem is low capacity of the column for two reasons:
1) I work with flavoproteins and once I wash the column, there tends to be a nice yellow strip on top of the column
2) when I measure activity on-line in flowthrough (i.e. when I start loading and at the end of loading), there is about the same activity (with only slight increase), so obviously it's already since the beginning that it doesn't bind.
What could I try to improve the binding to the chitin beads?
I don't have any experience with chitin affinity chromatography, but I will make two suggestions.
(1) Since the method is based on binding to a sugar, maybe you should leave out the glycerol, which is a sugar. At 5%, its concentration is over 0.5M.
(2) Try reducing the NaCl concentration. NaCl is mildly chaotropic and may therefore be interfering with the MBP structure. The datasheet for NEBs chitin beads recommends using 0.5 M NaCl.
https://www.neb.com/-/media/catalog/datacards-or-manuals/s6651datasheet-lot0121203.pdf?rev=30977aa6309b415cac0ed26d27531ed1
The manual does suggest flowing through the juice 4 times with gravity fed wide columns (I use PD-10 columns). Sometimes I'm lazy and I only do 3. This will, and don't think for a second otherwise, take one whole day, if not two. Anyways I typically have very good binding, by SDS PAGE. Note this is pTXB1 on the C-ter, though I doubt there is any major differences. My protein is a bit less fussy about temperature so I tend to do my flow at RT, which also gives you the benefit of higher flow rate. I don't have glycerol in my prep though here I disagree with Adam, I don't think glycerol competes specific binding with a polymer. I feel that 1M of salt is enough to repel non-specific, and glycerol will only reduce the flow rate. I wash with tx100 as well so those two would increase the flow rate even more. And try spinning your juice before loading very, very hard for an hour to really remove debris, or even pass it through a .4 or .2 filter afterwards, which would also increase your flow rate.
So my suggestions is really try to speed up your flow, and just load many more times.
Adam B Shapiro I never thought that glycerol could interfere with chromatography in such way. I may try it.
Ian Hu well, in my opinion it doesn't make much sense to re-load to column, because then the concentration is lower and there is already something bound, so the equilibrium would be shifted towards less binding, wouldn't it?
Nevertheless, this time I have loaded it on the column and left it there overnight.
If you think of a fast-on slow-off kinetics with less than ideal binding affinity, or some sort of conformational change causing enhanced affinity once bound to the bead, you want to flush a few more times to challenge off the non-specific bindings? Just guessing. I did try loading a fluorescent protein and indeed the first round bound the most protein, the extra rounds had almost visible impacts on the colour of the flowthrough. Or its just placebo effect. Anyways I do always load multiple times.
Also note that I think pressure is highly correlated to the binding, as many affinity columns are, which is why the manual also says load slower by gravity if you have less than ideal binding
Have you tried doing this at a low pH (7.4 - 7.6) with 500mM NaCl ? How much time do you incubate the protein after addition of cleavage buffer ? This plays a very major role in chitin preps.
No, I haven't tried lower pH, the buffer at pH 8.0 was kind of the standard in our lab and I didn't have really a reason to change it.
If you mean after addition of the DTT, I keep it over weekend at 4°C.
Basically i incubate for close to 16 hours and then start to elute out the protein. You can optimise your cleavage efficiency by looking at the IMPACT manual. The cleavage also depends on the residues present near the tag.
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