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The protein isoforms I am interested in comparing appear as distinct bands on the gel I have already run. I have an Excel sheet with optical density measurements I obtained using ImageJ; it looks something like this:

Lane    Iso1       Iso2       GAPDH
1       149.06     194.646    893.08
2       832.654    494.473    148.335
3       49.998     490.539    147.361
4       29.347     208.53     120.652

For the other proteins I've analyzed so far, I've been computing fold-change relative to my loading control, GAPDH, with the following equation:

Fold-Change = Log2(Protein/GAPDH)

I'm not sure how best to compare my isoforms to each other and considering the following two equations:

1. Fold-Change = Log2[(Iso2/GAPDH)/(Iso1/GAPDH)]

2. Fold-Change = Log2(Iso2/Iso1)/GAPDH

I've already computed both of these values for all my samples, graphed them and found that the resulting graphs look pretty different. Which of the two equations do you think is a better way to compare the relative quantities of these proteins?

Alternative equations are also welcome.

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  • $\begingroup$ Are your lanes replicates ? $\endgroup$ – WYSIWYG Oct 7 '14 at 16:29
  • $\begingroup$ No: each lane was loaded with a different sample. $\endgroup$ – Slavatron Oct 7 '14 at 16:34
  • $\begingroup$ Now see, the problem here is that in the first equation your GAPDH normalization factor is lost. So finally there is no normalization. $\endgroup$ – WYSIWYG Oct 7 '14 at 16:36
  • $\begingroup$ Could you explain more fully how the normalization factor is lost? $\endgroup$ – Slavatron Oct 7 '14 at 16:44
  • $\begingroup$ Oh, now I see that dividing the numerator and denominator by the same value cancels that value's impact on the final answer. $\endgroup$ – Slavatron Oct 7 '14 at 16:48
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In my opinion you should use this formula:

$$ \frac{\text{log}_2(\text{Iso}_1/\text{Iso}_2)}{\text{log}_2(\text{GAPDH})} $$

This will normalize the relative fold differences between the isoforms with the loading control- GAPDH.

Since both numerator and denominator are log transformed they are in comparable domains unlike the formula-2 that you mention in your question. This is just a modified formula-2.

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