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]).

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