It has been some time since I wrote here: multiple deadlines from my PhD programs and my coming soon trip to Torino, Italy, made me work more on the bench and less on the keyboard.
I would like to share with you some really cool stuff that got me quite excited, even after so many protein purifications sessions. Just to show you that every day we learn something new, and some days we learn for a week.
Rushing and getting a UV280 block
I will start by saying that all this resolution (double the meaning) started because I was in a hurry. Yeah, the #2 rule in science (after rule #1, “research what passions you!”) is “don’t rush science!”. I had a slot for the AKTA
purifier that day but had to purify an additional protein so I was tempted to inject some 20mg worth of protein on a 5/50GL anion exchange column. Now, the protein purifier within me said “Chen, you won’t get away it; the resolution will look like a big-wide block of UV280 chromatogram”. Of course I shushed my instincts and went along with the loading and then with the gradient elution. Of course, I did got some massive double-block peaks, all too close, all too high, and all too “what-the-hell-I-am-gonna-do-with-this-s***!”).See the image below to see for yourself (ugly, ain’t it?). Even without looking at any gel separation, it was clear for me that the before and after the column the sample is 99.9%, like, a TOTAL waste of time. Exactly what I didn’t need at that point (or at any other point in my PhD). I was looking at that chromatogram and thinking “Oh, darn it, that other protein will have to be flashed freezed coz I am not gonna get to it today!”).
Diluting, loading and One that turn into Two
OK, so I am stuck with 20ml of samples that I can barely split into two fractions, one of those contain my target protein. Thinking, I remembered that the problem of overloading is that any small increase of ion strengths leads to discharge of a mass of protein mix, losing control of resolution. However, If I will take that a fraction of those peaks that I think represent my protein, reload it on a fresh column bed and then elute, I will gain higher resolution power on a fraction of the whole lot. It’s like doing a zoom-in on a part of the whole image. Yeah, I know to some this will be so trivial, but still, I was quite enthusiastic to realize this fix. Take note, though, that is not the same as splitting the sample into multiple small injections as you end up purifying the whole protein mixture each time without gaining much resolution. Unintentionally, by doing a rough separation of the target protein from most of the protein mix, I could then take use of the column’s resolution capabilities.
So, making a long story short, I indeed diluted a selected fractions pool of the first peak, reloaded and eluted. And what do you know? I just got 2 out of the one single peak! Of course I could know that the first peak was composed of three main peaks mainly due to the 254nm reading, getting a less sensitive reading of the sample’s elution (this is why it is important to run your purification with several detectors, if possible). You can see the chromatogram below.
Of course, as it happens in many cases, the gel looked not that good – I had a mix of both proteins (with differing percentage of each population). This was realized to be digested forms of the protein (it was cleaved at it’s midst) so the whole samples were frozen with a big question mark on the box…
So, to sum up, if you ever stuck with a block of protein not separating after an IEC, you might want to dilute and reload a fraction. It worth the try!
Wana share some of your experience with advance chromatography tips or tricks?