Hi all,
I am trying to concentrate my purified protein using Corning 30kDa concentrators and my protein is about 78 kDa.
After elution I have 1.5 mg of protein total in my fractions but after few concentration steps until the desired concentration (4 mg/mL) I ended up with 0.5 mg.
I checked by nanodrop my flow through, nothing. Where can my protein be ? (on membrane ?)
Thanks,
Dimitri
Any possibility that you might be lossing protein during the centrifugation (protein is traversing the membrane due to a high centrifugal force) .. what speed do you use? The second possibility some proteins tend to precipitate upon increasing concentration or removal of salt (especially hydrophobic proteins). Its difficult to judge as several factors are likely to play a role.
All the best!
It would seem like your protein doesn't like concentration step, probably it precipitates or interacts with the membrane : you can try to let some buffer in contact with the concentrator once you have finished with this step : if protein gets stuck in membrane, you may see some traces of it back into the buffer.
Otherwise, try different centrifugation speeds, probably lower (is your protein a membrane protein??), otherwise try other concentration methods to see if you get the same result.
Thanks for your answers. I centrifuge at x12 000g as it is recommanded by the manufacturer. Unfortunately I observed through the light some precipate, so I guess i have my answer...
Dimitri Janin it is way too high i guess, especially if you have a membrane protein, try centrifuging at lower speeds, or changing the centrifugal devices
Most likely your protein is forming a gel on the membrane. With these devices the protein concentration becomes very high close to the membrane.
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