Tuesday 1 December 2009

How many molecules of X are there in a band on a Western Blot?

One of the questions I have often enjoyed setting in western blotting assessments, and that always seems to give problems, is...

"You have set up a western blot in which you wish to estimate the amount of GLUT4 per 3T3-L1 adipocyte (the cultured cell line used in the practical). You know that a 35 mm culture dish contains 2 million cells, and gives 1.5 mg total protein. You also have a recombinant stock of GLUT4 at 50 ng/ml. On your gel you load 15 µg of protein from the 3T3-L1 adipocytes and 1, 5, 10 and 20 µl of the GLUT4 stock. After probing with antibodies, you have a 55 kDa band in the 3T3-L1 adipocyte lane and in the GLUT4 stock lane. The band in the 3T3-L1 adipocyte sample has the same intensity of band as the 10 µl GLUT4 stock. How many molecules of GLUT4 are there per adipocyte?"

The chances are you are getting 'freaked out' by all the weird numbers flying around. The answer is to go back to basics.

For example:

If you had something with a molecular weight of 10, and you loaded 5 g on a lane, you would have 0.5 moles in that lane.

You know how many molecules there are in a mole (it is a constant that is the number of atoms in 12 g of carbon 12), so that constant multiplied by the number of moles would be the number of molecules in the lane.

Let's say one mole of something contains 100 molecules (which it doesn't; the real number is much, much bigger).

So, if you have 0.5 moles in the lane, you would have 50 molecules in that lane.

If you have another lane with a known number of cells in, say, 25, and that lane has the same intensity of staining as the lane containing 50 molecules, you could say that lane also has 50 molecules.
These 50 molecules came from 25 cells, so each cell must have 2 molecules.....

Now you can work it through with 'simple' numbers. All you need to do is to plug in the 'complicated' numbers in the question...

If you struggle with 'Science Maths' then you may like to look at Maths4Biosciences

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