Protein‐Protein Interactions Identified by Pull‐Down Experiments and Mass Spectrometry

Abstract

The aim of this unit is to provide a method for the identification of new protein‐protein interactions. Pull‐down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein‐protein interactions. When coupled with mass spectrometry, pull‐downs can be considered as the protein‐based equivalent of a yeast two‐hybrid screen. To improve the isolation of specific binding partners, pull‐down methods are described involving the use of cross‐linking, large‐scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state‐dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry‐based identification of interacting proteins are described, including specialized gel staining techniques, band excision, and in‐gel tryptic digestion. Data interpretation and the most commonly encountered problems are discussed.

P2X7 Receptor-mediated Scavenger Activity of Mononuclear Phagocytes toward Non-opsonized Particles and Apoptotic Cells Is Inhibited by Serum Glycoproteins but Remains Active in Cerebrospinal Fluid

“…Mass Spectrometry-The serum proteins in selected fractions after size exclusion chromatography (Superdex 200) were separated by 10% SDS-PAGE in non-reduced conditions. The gel was stained overnight with Colloidal Coomassie as described previously (19). The protein bands of interest were cut, destained, and tryptic-digested before analysis by matrixassisted laser desorption ionization (MALDI) mass spectroscopy.…”

The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons

“…Rat brain nerve terminal synaptosomes were prepared as previously described (25) and lysed in 5 mM Tris, pH 7.5, 150 mM NaCl, 1% (vol/vol) Triton X-100, 1 mM EDTA, 1 mM EGTA, leupeptin (10 g/ml), 1 mM phenylmethylsulfonyl fluoride (PMSF), and EDTA-free complete protease inhibitors (Roche). Conditions for binding of GSTtagged proteins to glutathione-Sepharose beads (Merck) and washing differed according to whether subsequent pulldowns were performed with bacterial lysates (24) or rat brain nerve terminal synaptosomes (26). Immobilized GST fusion proteins then were incubated with mixing at 4°C for 3 h with 1 ml of bacterial lysate containing His 6 -tagged protein or 1 h with 1 ml of lysate of rat brain synaptosomes.…”

Type VI Secretion Effectors: Methodologies and Biology

“…Therefore, the T6SS effectors were found to be directly or indirectly associated with Hcp, VgrG, PAAR, Tap-1/TEC, or Eag in various bacteria (Shneider et al, 2013; Silverman et al, 2013; Hachani et al, 2014; Liang et al, 2015; Unterweger et al, 2015; Whitney et al, 2015; Bondage et al, 2016; Cianfanelli et al, 2016a; Rigard et al, 2016). Because the genes encoding these conserved T6SS components functioning as carriers or chaperones/adaptors for T6SS effectors are easier to be predicted than most effector genes in a sequenced genome, these proteins can be first identified and used as a bait in well-established protein–protein interaction platforms such as bacterial two-hybrid (Battesti and Bouveret, 2012), yeast two-hybrid (Mehla et al, 2015), or co-IP/pull-down assay (Brymora et al, 2004; Kaboord and Perr, 2008) to identify potential effectors. This idea was indeed proposed by Silverman et al, that interaction with the Hcp chaperone may be a novel method to identify new T6SS effectors (Silverman et al, 2013).…”