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I am learning about western blot data quantification from some online resources. I have read about methods to normalise the data.

I have seen in a number of resources, such as this handbook and this video when normalising western blot data, they use a lane normalisation factor. For example, if using a total protein stain to normalise the integrated density of your target protein bands, they first obtain the integrated density of all the lanes stained with the total protein stain. Then they find the lane with the highest integrated density value. For each lane, its integrated density value is divided by the highest integrated density value - the resulting value being the normalisation factor of the lane.

Then when doing the normalisation, for each lane, the integrated density of the protein band of interest is divided by the lane normalisation factor.

However, I am not sure what is the reasoning behind calculating the lane normalisation factor. Why can you not just divide the integrated density of your protein band of interest by the integrated density of the total protein stain region for each lane?

Any insights are appreciated.

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  • $\begingroup$ Read point 2 of your handbook: II.TheIdealWorldvsTheRealWorld. $\endgroup$
    – KaPy3141
    May 17 at 15:30
  • $\begingroup$ Thank you, I think I understand now - it's to correct for variations in total protein signal between lanes. $\endgroup$
    – ceno980
    May 17 at 22:20

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