Hi,
I am trying to purify a protein from its endogenous source. The protocol recommends the use of Mono S (a strong cation exchanger). Can I use SP sepharose fast flow (which is also a strong cation exchanger) rather than Mono S? would it work fine and help in the desired purification?
I agree with Dominique. It should work. However, with these things you can't be certain until you try. Each protein has its peculiarities and each chromatographic support also has its peculiarities. We tend to view chromatographic resins in a one-track way - e.g. if it is a ion exchanger we tend to forget that the matrix itself might confer hydrophobic properties for example. So be prepared to play with conditions, pH, choice of salt etc. to optimise your own protocol.
Hi there,
It shouldn't be any problem as the cation exchanger is a sulfonate in both cases. The main difference being bead size (10µm for monoS and 90µm for SP Sepharose) and the chemical nature of the matrix (polystyrene for monoS and agarose for SP).
I agree with Dominique. It should work. However, with these things you can't be certain until you try. Each protein has its peculiarities and each chromatographic support also has its peculiarities. We tend to view chromatographic resins in a one-track way - e.g. if it is a ion exchanger we tend to forget that the matrix itself might confer hydrophobic properties for example. So be prepared to play with conditions, pH, choice of salt etc. to optimise your own protocol.
MonoQ and MonoS are expensive high-resolution resins owing to the small bead size. The columns are small and therefore have low capacity, so they are used where high resolution chromatography is needed but not high capacity. This would typically be at the end of a series of steps. For cruder starting materials, a cheaper, higher capacity resin such as SP Sepharose FF allows a larger column to be made, but the resolution is lower that MonoS.
Thank you Dominique and Paul. I will give it a try to see how it works for me.
Thank you Adam. As you said the use of Mono S is suggested at the end of series of purification step in the protocol I am following. Currently I am not having Mono S what if I use SP sepharose at the end of it??
There is no reason to think that it won't work. You will have to try it and see.
As an alternative to Mono S, you might try the Source 15S (or pre-packed Resource S) media for the same method. The bead sizes in the source media are slightly larger than the Mono media (15 um against 10 um) - but the intended use (final polishing step) is the same. Mono gives slightly better resolution, but it depends entirely on whether you need this. The Source media has a higher binding capability, and generates much less back pressure, than Mono - and the pre-packed columns are cheaper (list GBP 429 vs GBP 1,168 for a 1 mL column).
Might be worth thinking about?
SP Sepharose FF (average particle size 90 um) is usually used in early capture step, whereas SP Sepharose HP (average particle size 34 um) could be used in later polishing step. You can try SP Sepharose HP.
MonoS (10um) and RESOURCE S (15 um) have smaller particle sizes and of course their resolutions are much higher. They are more expensive and have higher pressure.
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