How the West(ern) was won: Solutions for immunoblotting large and small proteins
Relative protein quantification is a well-established technique in the vast majority of molecular biology laboratories. However, western blot standard protocols may not detect proteins of certain sizes. When the molecular weight of protein of interest is out of the 10–250 kDa range, its migration through the gel or transfer to the membrane is compromised, making its detection difficult. Here, we present a set of modifications of the standard working procedure for western blotting based on the experience working with small VCP-interacting protein and MAX-gene-associated protein, whose molecular weights are 8 and 350 kDa, respectively. We expect that these adaptations may help researchers to improve their experiments in a cost-effective manner.
- Blum A, Wang P, Zenklusen JC, 2018, SnapShot: TCGA-analyzed tumors. Cell, 173(2): 530. https://doi.org/10.1016/j.cell.2018.03.059
- ENCODE Project Consortium, 2012, An integrated encyclopedia of DNA elements in the human genome. Nature, 489(7414): 57–74. https://doi.org/10.1038/nature11247
- Iorio F, Knijnenburg TA, Vis DJ, et al., 2016, A landscape of pharmacogenomic interactions in cancer. Cell, 166(3): 740–754. https://doi.org/10.1016/j.cell.2016.06.017
- Llinàs-Arias P, Rosselló-Tortella M, López-Serra P, et al., 2019, Epigenetic loss of the endoplasmic reticulum-associated degradation inhibitor SVIP induces cancer cell metabolic reprogramming. JCI Insight, 5: e125888. https://doi.org/10.1172/jci.insight.125888
- Llabata P, Mitsuishi Y, Choi PS, et al., 2020, Multi-omics analysis identifies MGA as a negative regulator of the MYC pathway in lung adenocarcinoma. Mol Cancer Res, 18(4): 574–584. https://doi.org/10.1158/1541-7786.MCR-19-0657
- Llabata P, Torres-Diz M, Gomez A, et al., 2021, MAX mutant small-cell lung cancers exhibit impaired activities of MGA-dependent noncanonical polycomb repressive complex. Proc Natl Acad Sci U S A, 118: e2024824118. https://doi.org/10.1073/pnas.2024824118
- Lowry OH, Rosebrough NJ, Farr AL, et al., 1951, Protein measurement with the Folin phenol reagent. J Biol Chem, 193(1): 265–275.
- Azagra A, Meler A, de Barrios O, et al., 2022, The HDAC7- TET2 epigenetic axis is essential during early B lymphocyte development. Nucleic Acids Res, 50(15): 8471–8490. https://doi.org/10.1093/nar/gkac619