Yesterday was my birthday, and I tend to use this time in April to re-up my commitment to all of those resolutions that I failed at around Jan 5th.
And add a new one: Data is the name of the game.
- Millions of cells in the incubator? Check!
- Freshly-made solutions and buffers? Check!
- 10-15 Western blots/week? Check!
- Will to live? Fading... but still there!
There's nothing worse than this phase in a scientific process. I know the answer to my question and now I just need to generate a few figures that are presentable for publication.
That's a loading control for an assay called a western blot - it's a way to look at the protein content of cells. This is looking at a protein that should be present in equal quantities in all my samples. It looks pretty good, except that 5th lane is all askew, and there seems to be a bit less in the 3rd lane. So I gotta scrap the whole experiment and start over.
This is a recipe for frustration, and I've been banging my head against this problem for about 3 months. But I will get it. My new year's resolution at the beginning of my 28th year is to be diligent, be precise, and get some frackin' data.
Send me good thoughts, hopefully I'll be able to resume a regular blogging schedule soon.
I'm in the same place right now, but with gene expression data. Problems with inhibition... and as much data generation as humanely possible.
It's the worst.
If you have a loading control can you not determine the band densities in each lane and express them as a ratio to that control? Would not this take care of getting the same amount loaded into each lane?
Hi Jim - it's actually easier than that. I don't even have to run the gel first, there are ways of determining the total amount of protein in a sample ahead of time. And I do that, and most of the time it's fine. The trouble isn't always the loading control (actually, it rarely is), that's jus the easiest to show.
Loading the same total amount of protein can still give you wide variation in the loading control.
What would be neater is a preload assay that detects only the loading control.
Usually, the loading control is something like actin, which is usually found in the same proportion relative to total protein (in the same cell type). The goal is not necessarily to load the same amount of the loading control, the loading control is merely a read-out to show that you added the same amount of total protein in each sample.