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I have been running Western Blots on rat brain tissues from rats that have been subject to neurological disorders. I loaded the samples on the gel to ensure that for each tissue, I had a constant mass of protein (30 ug). The volumes were determined via BCA analysis. However, the BCA analysis was performed years ago (approximately 4-5 years ago). Also, the tissues have undergone multiple freeze and thaw cycles.

In addition to the tissue, I also loaded the same volume of Laemelli buffer as tissue and I used RIPA buffer to make the volumes of each sample constant.

I probed my membrane for my protein of interest and a housekeping protein (actin). I have attached a photo of my membrane down below.

enter image description here

top band is actin and bottom one is my protein of interest

However, I noticed that my actin bands are not consistent (some are fainter or darker than others).

Do the actin bands have to be the same darkness for each sample? Could I still use these results and instead do a normalization?

Or would I need to rerun this sample again?

Thanks for your help!

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The long and the short of it is that your actin bands do not need to be consistent so long as you can normalize and have met the quite stringent conditions for normalization to be effective. For normalization you generally need to have loaded less than 10 ug of protein, have not over-exposed any of the bands, and have them all loaded on the same gel and blot for comparison. You can not compare results across more than one blot without running inter-blot comparison controls!

Please note that this image would not probably not meet most publication requirements (in my opinion).

Please read the PDF here from Li-Cor on western blot normalization. It are the best descriptions of the conditions needed that I have come across.

However, for publication, you may have a harder time convincing reviewers that your results represent real data if the actin bands are so variable. Having a plot of normalized results is all well and good, but the eye is not very good at determining changes of less than about 50% (i.e. 2-fold) without careful consideration, so for instance your lanes 4 and 5 (not including ladder) have faint actin, but the POI is more or less consistent with the surrounding lanes - meaning that in 4 & 5, you have much more POI than in those other lanes, but it takes some thought to realize this.

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