Everything seemed to be going so well. All along the 6 week lab we enjoyed the warm green glow of our babies. Then came the time to seperate our babies from the undesirables by HIC. It's extremely hydrophic and should be washed off late as our salt concentration decreases we all agree. But what's this, there it is plain as day in the early fractions! We have no manual to follow this whole time. Just our instructor telling the 4 of us what to do from time to time. Here's the background. The HIC support resin is a few years expired but in a factory sealed bottle. The buffers used were 10 micro-molar potassium dihydro-phosphate for the low salt and we added KCl to the same to make it .5 molar high salt. The only few references I could find on this matter use ammonium sulfate buffers. These are the only parts of the lab that I can point a finger at. This is 300 level Biochem and most of these concepts are new at this point, but I'm learning quickly. Any feed back is appreciated. Thanks-
I should also mention that UV absorbance reading, molar extinction coeffecient, and Beer's Law revealed our sample contained about 4% GFP and alot of other junk that washed straight out of the column.