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,
Chris.
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