I have used Image Studio lite software, and it gives the respective intensities of the proteins. Can anyone guide me how to convert those intensities in fold change? I am working with wet transfer western blotting and chemilumiscence detection method to visualize bands after incubation with ECL reagent.
I do not know that Software, ImageJ is not enough? Using the appropriate control bands to normalize?
If you are going to quantify change by immunoblotting, you should also try several different loadings of your sample to make sure you are working within a range where changes in abundance cause a linear change in signal intensity
As Bruce Allen wrote, you can use loadings of different amounts of your sample to measure the variation in signal intensity with sample amount, thus creating a standard curve. The standard curve doesn't have to be linear, though. It just has to have positive slope so that there is a measurable increase in signal with an increase in sample. Importantly, the signals of the experimental samples must fall within the range of signals covered by the standard curve.
You want to know how to calculate the fold-change?
Take the intensity value for your 'control', whatever that may be. You will then divide the 'treatment' band intensity value by the value of the 'control' band. This will give you the fold change. If you want to show as '% of control' just times that value by 100.
Denstiometry is the best option. By comparing your samples with known control. Some free software are also available online.
I am not familiar with Image Studio lite software but the answer to your question really hinges on the way your are detecting the ECL reaction. The light that is generated is most often detected using film or a camera, such as in the Odyssey instrument. If you are using film, you must be aware that it has a dynamic range of only about one order of magnitude (10-fold) and most people misjudge what is a saturated region. In contrast, a camera can have a five log dynamic range and thus give better quantitation, or at least reduce the risk of saturation and incorrect conclusions. Absolute quantitation will require inclusion of a control of known amounts, as indicated above. Relative quantitation gives you fold changes and does not require having a pure source of antigen.
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