homogeneity of recombinant protein dilutions

[mtegg]

Hi, I've been having some problems with recombinant insulin-degrading enzyme, I have tried human and rat forms in a western blot and either nothing comes up at all, or the bands come up different on the duplicates and sometimes there is more than one band. I was thinking the diluted enzyme may not be homogenous, I am diluting in water and vortexing thoroughly before pipetting however.

The last one I did, I ran 7 serial dilutions of 200ng/well, down to 3.125ng/well, and only the 200ng band came up on 3 and 5 minute exposures, and I could just see the 100 and 50ng bands (which looked the same) on an overnight exposure. I thought that 5-50ng of purified protein was the standard range for western blots.

I have tried three different lots of the human IDE, and one of rat IDE, with inconsistent results for all. My internal standard always looks fine. Can anyone help me?

[danfive]

You have to be careful with recombinant proteins. They are usually low concentrations and in Tris or PBS. They are liable to freeze-thaw effect, cold-denaturation, pH drops (not in Tris), air-liquid & liquid-solid interphase degradation etc.

Proteins occur in Native state, Denatured state, Aggregated state.
The following equation explains the nature of the relationship:
1) Native state <===>Denatured state---->Aggregated state
Once aggregated the protein precipitates and disappears from solution and is worthless to the researcher.
The native and denatured states do have a sort of equilibrium that favors one or the other, depends on the protein and its environment (the solvent, temperature, presence of adjuvants or other proteins).

Normal precautions are to--
1) make aliquots to limit freeze-thawing
2) use soon after receiving.
3) add Tween20 to solution (~0.007%); non-ionic surfactant that protects from air-liquid interphase.
4) store in Tris, doesn't change pH with temperature.
5) store as concentrated as possible or in presence of BSA.
6) label with date of receipt, use within 1-3months.

[mtegg]

Thanks Danfive for your help, I will definitely look into adding Tween-20 and/or BSA and perhaps be a little more careful about thawing on ice. I appreciate your assistance.