Anybody that has used Microcon from Millipore to concentrate protein!? - (Apr/18/2006 )
Hi! I bought a kit from Millipore called Microcon which promises 100-fold concentration of proteins and DNA from 500microliter sample of my protein. I did everything as they said and the solution volume got reduced to 10microliter BUT my proteins did not get concentrated at all, the amounts look the same on the gel!
The thing is taht we want it in high concentration becasue we are intending of sending some bands to N-terminal sequencing. The problem is that I need to dialyse them before running on SDS gel because we use a relatively high salt medium for our bacteria. But after the dialysis the proteins are veeery diluted and we wanted to buy a kit that would concentrate them!
So any Microcon people out there? Any advice is more than welcome!
We have been using the microcon centrifugal concentrators without any such problems. Things that could potentially go wrong is damaging the membrane when applying the sample or using the wrong molecular weight cut-off for your protein of interest. Otherwise the only other thing I can think of is that perhaps your protein maybe irreversibly binding to the membrane.
Why do you think that would be the case, that it is irreversibly binding to the membrane?
are you doing that step at the end, where you invert it? that will pull quite a bit off the filter....
we also have had good luck with microcons...?
We have been using the microcon centrifugal concentrators without any such problems. Things that could potentially go wrong is damaging the membrane when applying the sample or using the wrong molecular weight cut-off for your protein of interest. Otherwise the only other thing I can think of is that perhaps your protein maybe irreversibly binding to the membrane.
Why do you think that would be the case, that it is irreversibly binding to the membrane?
Some proteins that have high proportions of hydrophobicity have been known to stick to membranes, although I have never encountered it. Its unlikely, but the only other thing I could think of.
Have you checked the flow-through for protein content or on a gel? If your protein has gone through the membrane then there is either something wrong with it or its the wrong MW cut-off.
Also, I assume you are using non-denaturing conditions? Using urea or GnHCl and the like could potentially allow flow through of your protein.
Thanks for all the comments! I am not looking only at one protein, I am looking fpr all the proteins I have in the supernatant as we are interested in N-terminal sequencing of the ones found in the mutants but not in our wild type. In any case, I've checked the filtrate and retenate and the filtrate doesn't give me any proteins and the retenate doesn't give me that much more concentrated proteins after 20 minutes of spin.
According to the protocol, I am supposed to get 10 microliters of retenate from 500microliters sample after 30 minutes spin, but I am getting almost 250microliter retenate aftef 20 minutes of spin! How is your yield?
We have been using the microcon centrifugal concentrators without any such problems. Things that could potentially go wrong is damaging the membrane when applying the sample or using the wrong molecular weight cut-off for your protein of interest. Otherwise the only other thing I can think of is that perhaps your protein maybe irreversibly binding to the membrane.
Why do you think that would be the case, that it is irreversibly binding to the membrane?
Some proteins that have high proportions of hydrophobicity have been known to stick to membranes, although I have never encountered it. Its unlikely, but the only other thing I could think of.
Have you checked the flow-through for protein content or on a gel? If your protein has gone through the membrane then there is either something wrong with it or its the wrong MW cut-off.
Also, I assume you are using non-denaturing conditions? Using urea or GnHCl and the like could potentially allow flow through of your protein.
Yes, it's non-denaturing conditions.
we also have had good luck with microcons...?
Yes, of course i invert it and spin down to collect the retenate.
According to the protocol, I am supposed to get 10 microliters of retenate from 500microliters sample after 30 minutes spin, but I am getting almost 250microliter retenate aftef 20 minutes of spin! How is your yield?
At the moment you appear to have gone from 500ul to 250ul which will result in a x2 concentrate. If you don't end up with the volume or concentration you require, try simply spining for longer.