Rab Antibody Characterization: Comparison of Rab14 Antibodies
Rab14 functions in the endocytic recycling pathway, having been implicated in the trafficking of the ADAM10 protease, GLUT4, and components of cell–cell junctions to the plasma membrane. It localizes predominantly to endocytic membranes with a pool also found on trans-Golgi network (TGN) membranes, and is most closely related to the Rab11 subfamily of GTPases. Certain intracellular bacteria such as Legionella pneumophila, Chlamydia trachomatis, and Salmonella enterica utilize Rab14 to promote their maturation and replication. Furthermore, the HIV envelope glycoprotein complex subverts the function of Rab14, and its effector the Rab Coupling Protein (RCP), in order to direct its transport to the plasma membrane. Since the use of antibodies is critical for the functional characterization of cellular proteins and their specificity and sensitivity is crucial in drawing reliable conclusions, it is important to rigorously characterize antibodies prior to their use in cell biology or biochemistry experiments. This is all the more critical in the case of antibodies raised to a protein which belongs to a protein family. In this chapter, we present our evaluation of the specificity and sensitivity of a number of commercially available Rab14 antibodies. We hope that this analysis provides guidance for researchers for antibody characterization prior to its use in cellular biology or biochemistry.
Key wordsRab14 Antibody characterization Immunofluorescence Intracellular transport Effector
We would like to thank Jacques Neefjes and Cecilia Bucci for their kind gifts of Rab14 and Rab7 plasmid constructs, respectively. This work was supported by a Science Foundation Ireland Programme Grant (09/IN1/B2629).
- 12.Kuijl C, Pilli M, Alahari SK, Janssen H, Khoo PS, Ervin KE, Calero M, Jonnalagadda S, Scheller RH, Neefjes J, Junutula JR (2013) Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria. EMBO J 32(5):713–727. doi: 10.1038/emboj.2013.10 CrossRefPubMedCentralPubMedGoogle Scholar
- 15.Romano JD, Sonda S, Bergbower E, Smith ME, Coppens I (2013) Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole. Mol Biol Cell 24(12):1974–1995. doi: 10.1091/mbc.E12-11-0827 CrossRefPubMedCentralPubMedGoogle Scholar
- 18.Qi M, Williams JA, Chu H, Chen X, Wang JJ, Ding L, Akhirome E, Wen X, Lapierre LA, Goldenring JR, Spearman P (2013) Rab11-FIP1C and Rab14 direct plasma membrane sorting and particle incorporation of the HIV-1 envelope glycoprotein complex. PLoS Pathog 9(4):e1003278. doi: 10.1371/journal.ppat.1003278 CrossRefPubMedCentralPubMedGoogle Scholar