Hello,
I washed the column with 0.5M NaOH and then 20% ethanol, then equilibrated with the running buffer, then injected my sample and did the purification.
After purification, I washed the column with the running buffer, then store it in 20% ethanol.
When I used again, I started washing the column with water to first remove the ethanol. Surprisingly, when I am pushing with a syringe from the bottom of the colum, the liquid that is coming out from the bottom is cloudy like it was the resin itself. Does anyone know what is happening?
Many thanks,
Julen.
Hi, it is highly unlikely to be resin.
It seems as though you washed the column only with running buffer (then 20% EtOH) after the previous sample? I think you are seeing material that remains in the column from that run. I'm not quite clear on whether the cloudy liquid is coming from the bottom or top - you say are pushing with a syringe from the bottom, so I think your cloudy material is actually coming out from the top? If so, I think it is most likely to be particulate/fatty material trapped in the upper frit or sitting on top of the column bed.
I would suggest you do a more thorough clean after the sample has been run, with high salt and/or 0.5M NaOH, but using reverse flow. Anything tapped in the upper part of the column is then likely to be flushed out before you load the next sample in the forward direction.
Superdex are spherical particles that you can distinguish by simple sight. Therefore, this does not make sense: "like it was the resin itself". It is or it is not.
Like Nick Gee says It's always best to wash out the column in reverse at a low flow rate rather than try to force all the contaminants through.
You should put a bolus of water between salts (even NaOH) and 20% ethanol, you may be seeing salting out.
OK, thank you so much. I will try to wash the column with high salt NaOH buffer.
Many thanks,
Julen.
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