While visualising cells stained with antibodies, I see certain proteins getting localized inside the nucleus or other organelle. However, when I perform subcellular fractionation and probe for these proteins in the western blot, I fail to detect them. My markers (like GAPDH, H3) for subcellular fractionation were detected in the correct fraction of cells.
Yes, you are right. Paraformaldehyde fixation can lead to the dissociation of protein-protein or protein-nucleic acid interactions in the cell cytoplasm as well as in sub-cellular organelles and lead to misinterpretation of the actual localization within a cell. For more detail see the link given below:
Paraformaldehyde Fixation May Lead to Misinterpretation of the Subcellular Localization of Plant High Mobility Group Box Proteins. PLoS ONE 10(8): e0135033. https://doi.org/10.1371/journal.pone.0135033
Article Paraformaldehyde Fixation May Lead to Misinterpretation of t...
Hello Harsh Oza
It is known that redistribution of proteins can occur if the fixation procedure does not completely immobilize the proteins, especially when fixation is done with PFA alone.
Please refer to the article below for more details.
https://pubmed.ncbi.nlm.nih.gov/10213201/
Therefore, if the protein under study is sensitive to fixation, it is recommended that PFA be used in combination with other fixative to be certain that the proteins are truly immobile and have not altered in organization due to fixation. Also, the antibody concentration, time of incubation, fixation and permeabilization and temperature of incubation can all affect protein distribution. So, all these parameters need to be optimized.
Best.
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