Hi everyone, I have purified my bacterial protein weigh 22kda after purification I start concentrating it but after concentrating a few ml my protein start precipitating aven I have kept my ph 1 unit above the Pi of protein. Any suggestion?
Dear Asmat
1 ph unit from the pH is not a lot. you can try to use also buffer with more different pH., because the theoreticall pI, which is calculated on the linear protein sequence may differ a lot in some cases from the real pI which is depends from the protein surface charge/hydropobicity if the protein is folded.
However to provide a reliable answer to your question i need to know some more details about the purification approach that you use, the buffer and how much the protein was concentrated when the precipitation happen. Did your protein contain cysteines and or hydrofobic regions?
best regards
Manuele
i am trying to exchange the elution buffer having 500mM imidazol and 200mM phospate and 2mM dtt with 20mM imidazol free buffer when my sample volume reduced from 30 ml to 10 ml it strts precipitation even i am not been able to get my required concentration of 1mM protein for NMR structure sample
Dear Asmat,
To elaborate on Manuele Martinelli's answer.
The ionic strength and pH are very important parts of the equation, and indeed 200mM Phosphate gives on its own a quite high ionic strength. If you directly go down to 20mM phosphate without any salt to compensate that sudden 10-fold drop in ionic strength, that could in itself already cause issues. Just in case : a quick check of protein stability in various buffers could help finding more optimal conditions (see e.g. 10.1002/0471140864.ps2809s79 and https://www.biozentrum.unibas.ch/fileadmin/redaktion/05_Facilities/01_Technology_Platforms/BF/Protocols/Preventing_Protein_Aggregation.pdf)
The question of reducing agent Manuele mentioned is also important, though as it was generously present in your starting buffer, lack of it is less likely to cause a strong precipitation within the relatively short timescale of a concentration.
The method of concentration can indeed also be quite sensitive. In the widespread centrifuge conical filters (that I would assume you used), the protein tends to accumulate near the filter, creating quite high local concentrations at the bottom of the funnel which (if the buffer is not optimal for the protein) could trigger precipitation. I would suggest in this hypothetical case to :
- frequently resuspend your concentrate by pipetting to prevent this accumulation
- change your concentration method e.g.:
- ultrafiltration using pressurised gas, which is typically done in stirring cells that prevent such accumulation on the membrane.
- using a centrifuge filter where the membrane is above the protein solution (on a plunger) thereby preventing that strong local accumulation.
- though requiring high quality salts and some preliminary calculations, you could try a dialysis against your final buffer supplemented with high-purity high-molecular weight PEG, which when osmotic pressure is gentle enough is a very soft way of concentrating while buffer exchanging
- there are methods of native precipitation of the protein (e.g ammonium sulfate) but I would maybe skip on that in an already aggregation prone protein
- just in case, even though I am pretty sure you didn't overlook that, double check that your membrane cutoff is much lower than your 22 kDa protein.
Finally : there are proteins that unfortunately don't stand millimolar concentrations. In the unfortunate case yours would also behave so: try to very progressively concentrate part of your sample, regularly checking attained concentration, until precipitation occurs. You would then know where you need to stop. One can still record good multidimentional spectra - including NOESY - on a sample of only a few hundreds of uM.
good luck!
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