[rulba]

Hi guys,

I have this simple question. I am going to reconstitute some protein. I know it Can precipitate due to denaturation if It gets in a concentration above 20 mg/mL. I have 1 g in a 2 mL container. Should I, in a laf bench, transfer the protein (say 100 mg) to a nalgene bottle containing 6 mL sterile water instead of doing it the normal Way and transfer the water to the vial with the freeze dryed powder? However if it has this solubility, I have difficulties in understanding the final volume of the container containing the freeze dryed powder. It must have involved moving it after sublimation of the water?

Thanks in advance for your time.

Best regards

[Arkcon]

Its hard to follow what you're saying.  If I understand you, you're worried about the protein denaturing because the concentration is too high.  Probably what you mean is aggregating, and losing some activity.  So now, you're debating how to mix it with water, I have to guess, because for a few seconds it will be too concentrated?  But water to solid or solid spiked on water -- for a few seconds, it will always be the case that the mixture will be very concentrated.  So it shouldn't matter, or if it does matter, there's nothing you can really do.  Right?  If this is a problem, you will have to address it some other way.

[rulba]

Sorry for my bad explanation. Yes I am afraid for aggregation, due to a too High concentration. So I was thinking if I dissolved a bit in the sterile water, I would avoid the High concentration. Right now it is in a small container containing one g. SE hplc indicates that solubilities above 20 mg/mL causes some hmw aggregates.

Thanks in Advance

[Babcock_Hall]

Was the protein originally lyophilized in pure water or a buffer?  Do you have any flexibility in terms of the solvent that you use to reconstitute the protein?  In other words, do you have to use pure water, or can you choose a buffer?  The pH of a solution is a key variable in how soluble a protein is, and ionic strength is often important as well.  In any case, I think that carefully weighing out a small portion of the lyophilized powder and trying to reconstitute that portion alone makes sense.  If something goes wrong, you can change your technique for the second batch.

[rulba]

Thanks for your reply.

It was lyophilized when I Got it in sodium phosphate buffer (ph 7,4). We are going to run a 1D SDS gel, so I Will try to dissolve a little in some sterile water and make the analysis.

Thanks alot for help.

[Yggdrasil]

I would advise against dissolving in pure water because, as Babcock_Hall has said, having the proper pH and salt concentration is essential for maintaining proper protein structure and function.  I would recommend using dissolving in something like PBS (phosphate buffered saline).  That should not interfere with most downstream applications.

Of course, if all you're going to be doing is running a SDS-PAGE analysis, it doesn't really matter if your protein denatures or aggregates during reconstitution as you'll be denaturing your sample anyway to run the SDS-PAGE gel.

[rulba]

Thanks for your reply. It was lyophilized from a buffer, so if I dissolve it in sterile water it should remain buffered. We Will also run a maldi ms, but I would additionally Like to determine which solvents that causes less aggregation by running se-hplc and activity asssays, so I am going to try different buffers.

Best regards