Hi everyone,
I've immobilized different Abs to NHS-activated agarose resin. Coupling process was OK.
When I add different samples to my column, Abs capture their cognate antigens. The problem is, when I try to elute all the Ag (or as much as possible), a great percentage of the Ag is retained into the column.
Nowadays I'm using an acidic buffer (Glicine pH 3) to perform elution. I tried with other types of buffer (high pH and Ionic strenght) but I didn't work. At least, with low pH I recovered part of the Ag. I also tried different time and temperature conditions, but the results were the same.
Any suggestions to improve my results?
Thanks in advance,
Florencia.
Try to electroelute the bound protein from the column, there are commercial apparatus available for cut out bands from electrophoresis, these work with affinity gels too. This allows more complete recovery under milder conditions. If all else fails, add denaturants like urea, but then you may not be able to reuse the column.
I think you can make progress with minor revision to your protocol. pH 3 is not low enough for most antibody affinity columns. Use glycine buffer at pH 2.3. There are two benefits, (i) more forceful elution (ii) more abrupt transition from equilibration pH to pH 2.3, because glycine pH 2.3 is operating near its pKa value. You'll need to neutralise the eluted fractions in the normal way. If you are concerned about the impact of pH 2.3 buffer on the antigens, I'd collect small elution fractions in eppendorfs and neutralise each one in turn, rather than all tubes at the end..
Hello Engelbert and Nick, thank you for your helpful answers.
I've tried using my buffer at low pH (2.3 to be precise). Also, I proved with the addition of a mild detergent (instead of using urea). Unfortunately, I still have problems with elution process. Almost all the Ag is retained on the column.
Anyway, I will keep trying! Suggestions are always welcome.
Are you certain that the antigen is actually binding to the column (i.e. absence of antigen in the flow though material)? If that checks out, it is possible that you have an extremely high affinity interaction which is hard to break.
Or, you are recovering more antigen than you think. How are you measuring recovery? Are you measuring protein or biological activity (which may have been compromised)?.
I am going to assume it's an affinity issue, in which case you could try attaching less antibody to the column to reduce the risk of rebinding after elution. However, an easier option if you are not trying to preserve biological activity would be to lower the pH even more, though this is not normally needed.
As Englebert says, you could add denaturing agents but your column may then be single use.
Another option is to elute with high pH (pH 11).
Hope you have some success.
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