'Does this mean I'll glow in the dark?'

Herein marks the beginning of my final week in the lab. The eight weeks have completely flown past and I can't believe it's coming to the end-- I feel like I should only be halfway through! Even scarier a thought is that in 3 weeks time I will be starting my third year and my dissertation... Cripes.

So last time I updated, there were question-marks here, there, everywhere. There is still no definite resolution to any of those questions yet-- but work has begun to solve the mystery. Because there is a lack of Zta banding in the blots (which is our positive control band) this raises questions as to whether something in the assay causes its' degradation or whether there was something wrong with the nuclear extract I was using. The solution to this is to create radioactively labelled Zta and run it through the procedure and see if it then bands (if it does, it means the nuclear extract had degraded). If not then there is something within the assay that causes this problem and the assay can be dissected to see where the degradation occurs.

After all this, before running the procedure again, the radioactive Zta can be doped in to nuclear extract to see that the modifications have worked. Hopefully then the next individual who tackles the project can get straight to work on isolating proteins (if there are any).

Of course, it's easier said than done because firstly we have to produce the radioactively labelled [S35Meth] Zta and I'm not trained to work with radioactivity. (Admittedly the idea of working with radioactivity brings up all sorts of 'Hulk' ideas in my mind and I'm not sure I want to mutate myself yet.) The first step is to generate this radioactive Zta-- through an in vitro transcription and translation system. This requires constant testing at every stage and it quite long and laborious... But it does look like we are producing Zta mRNA which is exactly what we need at this point in time.

With this being a long process, I've been helping out with some Maxi-Preps at the same time. And it's official: bacteria smell bloody funky. I felt like I could smell it on my skin for hours later and I kept getting really worried that customers at my other job could smell it on me too (I go straight from the lab to my other job some evenings). Anyway, I've managed to maxi-prep 6 separate colonies of bacteria with different mutations-- so I think I'm adjusting to the smell a little bit.

I've also done some mini-preps and restriction digests to compare different mini-prep techniques to see which generate the best results.

So yes, the last week has been spent doing bits-and-bobs around the lab whilst waiting for the next part of my own experiment to come into fruition. This week, of course, I have to start writing my report to go alongside my 8 week experience. Slightly daunting, but obviously everyone will be able to see how that goes when they get published.

Giant question-marks hanging over our heads...

I've got two weeks worth of work to power through, as I didn't update over the bank holiday weekend (which has nothing to do with laziness-- more to do with the impossible logistics of updating whilst moving house). Probably best to get cracking then!

As I said in my last post, I had all my samples prepared and ready to run on the SDS-Page and Western Blot for the beginning of the week. I had 6 samples in total:

-OriLyt 3 DNA with Nuclear Extract 1

-OriLyt 4 (negative control) DNA with Nuclear Extract 1

-OriLyt 3 DNA with Nuclear Extract 2

-OriLyt 4 (negative control) DNA with Nuclear Extract 2

-OriLyt 3 DNA with Nuclear Extract 3

-OriLyt 4 (negative control) DNA with Nuclear Extract 3

First I set these samples to run through SDS-Page-- a procedure used to separate proteins out in order of their molecular weight. The procedure should allow us to separate all the proteinous elements in a Nuclear Extract that bind to our OriLyt DNA, therefore giving us an idea of what elements associate with the origin of lytic replication (giving us the replisome of EBV).

After the SDS-Page was finished, I then had to transfer the separated proteins from the gel onto a nitrocellulose membrane for Western Blot analysis. This process is sort of like running an SDS-Page almost, it uses the same idea (running a current as a means of development) but in this process you create something dubbed the 'transfer sandwich' continuing the trend of 'bizarre items/names in science'.

Once the transfer has finished, I then had to block the membrane before amplifying the proteins using antibodies-- or, more scientifically put, I then subjected my membrane to a Western Blot. This was then developed via ECL which, this time, I got to make up myself.

Making up the ECL made me feel a little like a crime scene detective-- as ECL involves Luminol. For those who haven't been brought up on CSI like I have, Luminol is a chemical that is able to fluoresce blood and so is used to detect where blood traces have been (though the visible evidence has been removed). It makes a lot of sense that this is a key component of the ECL reagents that allow you to develop Western Blot samples.

When we developed the blot the first time, we could see that there were very weak bands appearing (figure 1 below). The problem with this is that the bands only appeared in the lanes containing OriLyt 4, NE1 and OriLyt 3, NE3-- so why did they only develop in these regions? And even then, what were they because they definitely didn't appear in the region we expected them to.

  Figure 1- Western Blot 1, with the two bands highlighted.

When I showed these results to my supervisor she was just as baffled as I was, so we decided to reblock the sample and reprobe it again to see whether it was a definite result. When we reprobed it, we got a much clearer blot (figure 2 below) which showed the same results-- but also showed clear bands in OriLyt 3, NE1 and OriLyt 4, NE 3 as well... Further adding to the mystery.

Figure 2- Reprobe of the original western blot, showing further bands. Green arrow indicates where bands should theoretically be found for Zta.

That brings us neatly around to what this week was about, although I've been a little ill and so I've been in-and-out of the lab. We decided that the lack of bands in our nuclear extract lanes could potentially be caused by it being too weak to be detected by the ECL-- therefore we decided to scale up the experiment (by eight).

Repeating the previous procedures we then got yet another mysterious western blot... But, the bands that have developed have once more developed in the same place as previous-- indicating there is something there (opposed to procedural error). Of course, there is still a giant question-marks hanging over us as we try and figure out what it is and why it is there (one of my fellow lab-peers has laughed and said that maybe I've discovered another form of Zta; that's wishful thinking and a half).

Figure 3- Amplified western blot using NE3. Shows bands in OriLyt 3 (right highlight) and a weaker band in OriLyt 4 (left highlight). Question marks are bands in which the appearance was previously dismissed as beads-- but cannot be anymore as there were no beads added to these samples (Nuclear Extract 3- far right; Supernatant 3- right; Supernatant 4- left).

So right now, we have more questions than answers.