Friday, 31 July 2009

Red, green and blue…

I promised you pictures of more western blotting in my last blog; I’ll confess now, I’m breaking that promise. Truthfully I don’t know where on earth I’ve saved them on my computer, but they’re very boring anyhow so I’m not going to waste my time or bore you by looking for them.

However, I do have a far more interesting and stunning picture for you in my opinion, which I took this week using fluorescent imaging …

In simple terms this is a picture taken of lots of cells, each cell’s nucleus is stained blue. The red is where I’ve stained the “scaffolding” (Actin) of the cell which gives it its structure and the green is a dye which has stained the zinc in the cell- which is what I’m studying.

I don’t think I can go into anymore details of the experiment or the picture you can see here as it may lead to something that can be published.

As you may have guessed, I have been mainly doing Fluorescent microscopy this week, which makes a nice change and has produced some pretty pictures whilst providing us with some definitely good results.

I’m going to do some more fluorescent imaging next week, and then FACS the week after to obtain quantifiable data which will support the fluorescent images I have taken. I’ll probably end up doing another few blots too, just to test some other molecules involved in the zinc wave and further support our theory.

I probably only have about three more blog entries to go, so keep reading!
I’ll hopefully some more fun pictures for you again next week.

Keeping this one short and sweet.

Peace out,

Your mad (but very happy with this weeks results) scientist, Chris.

Thursday, 23 July 2009

Never mind swine flu… yeast infections are rife the lab…

Luckily this week we put our usual order of cells on hold, as we needed to analyse the data from the previous immunoprecipitation experiments before making a decision on what to immunoprecipitate with next, but also we had a few samples left in the freezer to use from previous experiments. I have heard that other staff in the lab have had their samples infected with yeast, unlucky and frustrating for them, but a bit of a forewarning to me- don’t brew my own beer or make bread whilst growing up cells.

I’m actually HALF way through my placement now, and I can honestly say I feel like I know Western blot like the back of my hand, and probably immunoprecipitation and other bits and pieces surrounding running gel electrophoresis for Western blotting too.

I learned Densitometry this week, including how to photograph my blots to calculate the relative amounts of protein present in each band after quantifying the blots using actin or ZIP7 as our primary antibody, for normal and immunoprecipitated blots respectively. Also I’ve been using Paint Shop Pro software to enhance and clarify my images for presenting in a final project.

No fun pictures of me doing lab work this week I’m afraid, so I’ve put some examples of the blots I’ve developed (can’t describe in detail as it may need to contribute to a publication), as I know people generally prefer to look at a picture instead of read my rambling on. Not a lot of big news this week, but that usually comes out on a Friday when we’ve had our weekly meeting and tied up loose ends so I’m sure I’ll be updating you on that in my blog next week (which I will publish as normal on late Friday afternoon). Also I will have plenty of photographs in my next blog… I think I’m going to do it like a diary… “A day in the life of me” kind of thing.

Here are some pretty pictures in the mean time for you to ponder over.

Above is some Nitrocellulose paper, which has been stained with Ponceau S red dye to show up the proteins that have been blotted from my gel, to the paper.

And this is a photograph which I developed in the dark room, showing up some bands of an antibody I have used, which I've stained with a Chemiluminescent reagent in order for my antibody to glow in the dark.

Hope you enjoyed this post and are looking forward to my blog next week, which WILL be full of pictures.


Friday, 17 July 2009



I should be king of the Western blot by now, yet it continues to baffle me with the variation of the results it produces, even if I have treated two things exactly the same, they will seldom be the same. Last week I had problems with spots on my developments- so I altered my method of applying the chemiluminescent reagent, yielding pretty impressive-looking photographs. This week I have freckles, not spots- there are more and they are smaller; it’s as if some one has flicked ZIP7 over my nitrocellulose with a paintbrush. These freckles are unlikely to be caused by the reagent. Lindy was kind enough to share her western blot brochure with me, containing information about why things may not turn out as you hope with the western blot- it told me that these freckles could be either due to my antibody being too concentrated and binding to the milk block or perhaps my milk wasn’t mixed well enough and so on…

Today, I’ve been probing and developing blots for various things, my freckles weren’t as bad today when I had re-done the same experiment as I moaned about above. That’s Western blotting for you.

I didn’t take many pictures this week, as I find it slightly embarrassing asking people “can you take a photo of me doing this… can you take a photo of me doing that?” So here’s one of me harvesting my Tamoxifen-resistant cells that I had exposed to zinc for different periods of time in order to immunoprecipitate with ZIP7 to carry out various gel electrophoresis and western blot experiments.

I’m still really enjoying myself here, and I am still enthusiastic and happy to be working on such an interesting project. I haven’t written as much this week as I have generally been repeating experiments but I’m hoping I’ll have some good results to talk about next week. Also it’s my birthday next Friday, so I will be publishing next week’s blog entry on the Thursday, as I intend to go straight to the pub after work and not the IT lab! :p

Thanks for reading my fellow mad scientists,


Friday, 10 July 2009

1 in 50,000 – Dilution or Chance of getting a result with my antibody?

I didn’t know whether to change this title to “A rather exciting end to the week” as low and behold, I have just got some promising blots developed on a Friday afternoon after a week of what felt like it wasn’t meant to be! I thought I’d save it for next weeks blog though as I’m not going to be following it up until Monday. So keep reading!

First of all I have to say that I can’t believe that two weeks have gone already! I feel I’ve settled in quite well this week, and I’ve had a much better idea of what I’ve needed to do myself in order to get the results we want. Although I must give credit to Nicola, Jan, Lindy, Ed and of course Katherine for helping me out when I’ve messed up on a particular solution or couldn’t get my head around how much of what needed to go with so much of something in order to get the correct dilution.

This week, I’ve again been running gel electrophoresis and western blotting- from culturing new cells (some of which I will be immunoprecipitating with on Monday), to incubating with ZIP7 Rabbit antibody. I run 4 blots, the first two I used one reduced loading buffer and one non reduced loading buffer, to see if the ZIP7 formed high molecular weight molecules as a result. However, the 1 in 50,000 dilution of the ZIP7 antibody that I used (which worked last week), did not yield any bands, even though when the blot was stained with Ponceau S dye, many bands appeared. To begin with I thought the lack of bands was due to only incubating with the primary antibody for one hour, so I reapplied more 1 in 50,000 antibody and left it to incubate overnight, only to find the next day that again I had no results when I’d developed with Femto (a chemiluminescent reagent). Then I thought I wasn’t getting results as I hadn’t loaded enough sample onto the gels to begin with, so loaded another two gels in the same conditions but with three times the amount of protein, by this time I had worked out that it was probably something to do with the antibody (which turned out to be correct) so I made some new 1 in 10,000 primary ZIP7 rabbit antibody and incubated my new blots with this. I have just developed these blots- hence the excitement at the beginning of the blog, but I’m sticking to my guns- you can wait until next week! I’ll also be doing another technique next week- immunoprecipitation which I’ve never done before, so I’m eager to learn how to do it, and equally if not more importantly, I’m eager to get results which we would like to see!

Also today, the group working on the same project met together with Dr. Katherine Taylor (our supervisor in charge of the project), to discuss what we’ve done this week and what we’ve found; to keep everyone up to date on the goings on in our research area. I found it very interesting learning how another student - Makayla (working on a masters project) had been mutating specific genes in the breast cancer cells (which I’m obligated not to mention), so that I can then work on to test out a theory of how ZIP7 works and hence a possible route to control its activity.

All in all, I’ve had a lot to think about this week, and have another lot to think about over the weekend. But I still love the work and am happy and glad to be doing it!

Stay tuned kids! And I half promise to put pictures in here next week, ok, actually I promise, because if push comes to shove, I’ll just put one of something relatively amusing which would be a lot more effort!

Have a good weekend! You deserve it if you’ve just managed to get through all of this!

Chris. (not in the lab[right now]).

Friday, 3 July 2009

Time flies when you’re having fun…

I can’t believe I’ve been here a week already! It has gone so quickly and I cannot begin to explain how much new stuff I’ve learned already!

First of all, I have to say that I am thrilled that I’m working on this particular research project, as it’s completely fascinating and the group of people I’m working with are lovely and very helpful. I really love the atmosphere in the labs.

On Monday, I began my placement with two other students – Esther and Charlie. We spent the first two days with a PhD student –Rhiannon, who very clearly taught us how to separate proteins using the Western Blot technique. I was glad that I wasn’t expected to know what I was doing as soon as I got there, which was my main fear before I begun. Also the fact that I wasn’t the only new student relieved some of the pressure, as we’ve all helped each other out when we couldn’t remember where things were kept or what ingredients we needed to use to make buffer solutions for example.

Techniques I’ve been learning this week centred around Western blotting. First of all I harvested Tamoxifen resistant breast cancer cells which were already being grown for me. Then I lysed them and spun them in the centrifuge to separate the proteins we are interested in from the cell membranes which I do not need to use for this particular experiment.

Once the cells were prepared I performed a protein assay to quantify the amount of ZIP7 (which is a zinc transporter which we are interested in, involved in Tamoxifen resistance in breast cancer cells) in each cell batch, which were treated differently by exposing them to zinc for different lengths of time. Then I moved onto performing a complete Western blot (from making the gels, buffers, running the samples, transferring to nitrocellulose, incubating with antibodies and then developing the blot in the dark room and so on) where I probed the nitrocellulose for ZIP7 and various other proteins in the cell to yield some conclusive results.

What I have learned very quickly, particularly from the Western blotting technique is that science is unpredictable in many ways, and procedures can produce unclear results at any stage as a result of random or human error.

Also Dr. Katherine Taylor gave us a lengthy talk today about her published and new unpublished research which was very informative and extremely interesting.

Already towards the end of the week and today, I feel like I’ve been able to think logically and independently to carry on with work without much guidance.

Biggest challenge of the week- trying to remember where everything is kept!

All in all, I’ve enjoyed this week lots, and am looking forward to next week.

I’ll update you next Friday on any shenanigans or break-throughs (hehe) I may make!

Until then, peace out zinc lovers :-)