I am doing Western blot data analysis where I have images from a number of experiments (where the samples in the experiments are biological/technical replicates). For each experiment, I labelled the membrane with antibodies against the same proteins of interest.
I want to combine the data from these experiments so that they are all displayed on the same graph.
I took my images at a variety of exposure times (e.g. 5 sec, 30 sec, 1 min, etc). For one protein X, I have found that the images taken at 30 seconds exposure are the best for analysis, as the bands are saturated at higher exposure times.
However, for one experiment, for the protein X labelling, I cannot use the 30 second exposure image for my analysis as there are ghost bands. I have an image taken at 5 seconds exposure which I can use instead.
I was wondering when combining data from western blot images from different experiments, for a specific protein of interest (e.g. protein X), do all the images have to be taken at the same exposure time? All the protein X images I will be using were taken at 30 seconds exposure except for one which is taken at 5 seconds exposure.
Any insights are appreciated.