Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins <em>in vitro</em>

Abstract

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights.For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2 . In this assay, non-fluorescent N-and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins' interactions, the fluorescent signal is reconstituted [3][4][5][6] . Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3 . This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions 7 .Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport [8][9][10][11][12][13][14] , and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15 , is demonstrated using this technique. Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=2961 Differentially tag the proteins to be tested for their detection. Label the protein to be immobilized on the membrane (ProIM) with a tag of a larg...

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

“…Next, we confirmed that Pyk2 could specifically and directly bind to MCU and phosphorylate MCU by in vitro binding (72) and kinase assays (37) using purified MCU and Pyk2 proteins (Fig. 3A, B).…”

“…For electrophoresis under nondissociating conditions, mitochondrial proteins or whole cell lysates were separated by 10% polyacrylamide gel (Native-PAGE) (63). IP (37), in vitro binding (72), and kinase assays (37) were performed as described previously. (49), measurement of mSOF by mT-cpYFP (73), detection of caspases 3/7 activity (48) were performed in live cells using the laser scanning confocal microscope (Olympus, Tokyo, Japan).…”

Immunoprecipitation

“…An immunoprecipitation, commonly referred to as an IP, commences by addition of a specific antibody to a prepared cell lysate, allowing for the formation of antigen-antibody complexes (Taylor and Best 2011;Ueki et al 2011;Alexandru 2012;Cho et al 2012;Conlon et al 2012;Jain et al 2012;Poulsen et al 2012;Schmollinger et al 2012;Smaczniak et al 2012;Spiro 2012;Becker et al 2013;Cordeiro and Bidere 2013;Li et al 2013;Sarkar et al 2013;You et al 2013). Upon completion of the incubation period, Protein A, G, or L agarose or magnetic beads are added.…”

Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase

“…MPs modulate PD structure by increasing the size exclusion limit (SEL) through severing actin filaments or degradation of callose around PD, either directly or by employing host proteins to facilitate movement [ 4 – 8 ]. Viruses from the genus Potexvirus have their MPs organized as a triple gene block (TGB) and are speculated to target PD by endoplasmic reticulum (ER) association [ 9 – 11 ].…”

“…Viruses from the genus Potexvirus have their MPs organized as a triple gene block (TGB) and are speculated to target PD by endoplasmic reticulum (ER) association [ 9 – 11 ]. Various studies using the yeast two hybrid system and far western blotting revealed association of many host proteins with viral proteins: DNaJ family proteins of Nicotiana tabacum and Arabidopsis thaliana with the non-structural protein m (NSm) protein of Tomato spotted wilt virus (TSWV) [ 12 ]; multiprotein bridging factor 1 (MBF) from tobacco with MP of ToMV ( Tomato mosaic virus ) [ 13 ] and coat protein-interacting protein-L (IP-L) from tomato with the coat protein (CP) of ToMV [ 14 ]; pectin methylesterase (PME), calreticulin and ankyrin repeat containing protein from tobacco with MP of Tobacco mosaic virus (TMV) [ 8 , 15 , 16 ]; KELP from A . thaliana with MP of ToMV [ 17 ]; Rubisco small subunit from N .…”