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.