Protein‐Protein Interactions Identified by Pull‐Down Experiments and Mass Spectrometry
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
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.
Keywords: Pull?down; protein?protein interactions; small GTPase; GST fusion protein; mass spectrometry; spin column; cross?linking; in?gel digestion; tissue lysate
Table of Contents
- Strategic Planning
- Basic Protocol 1: The Pull‐Down Experiment
- Alternate Protocol 1: Effector Isolation with GST‐Tagged Small Gtpases
- Support Protocol 1: GST Fusion Protein Purification
- Support Protocol 2: Cross‐Linking GST Fusion Proteins to GSH‐Agarose
- Support Protocol 3: Preparation of Large‐Scale Tissue Lysates
- Basic Protocol 2: Sample Preparation for Mass Spectrometry
- Support Protocol 4: Recycling of GSH Agarose
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol 1: The Pull‐Down Experiment
Materials
Alternate Protocol 1: Effector Isolation with GST‐Tagged Small Gtpases
Support Protocol 1: GST Fusion Protein Purification
Materials
Support Protocol 2: Cross‐Linking GST Fusion Proteins to GSH‐Agarose
Materials
Support Protocol 3: Preparation of Large‐Scale Tissue Lysates
Materials
Basic Protocol 2: Sample Preparation for Mass Spectrometry
Materials
Support Protocol 4: Recycling of GSH Agarose
Materials
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Figures
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Figure 17.5.1 Flowchart for strategic design and the experimental processes described in this unit, showing how the planning and analysis stages (left‐hand side; a summary of the Strategic Planning section) are linked with the experimental protocols (right‐hand side). It is also a reference for how the protocols relate to one another. View Image -
Figure 17.5.2 The spin column–based pull‐down experiment. An illustrative description of the approach taken when an entire pull‐down experiment is performed within the spin column. This differs from the protocol presented in and the Alternate Protocol, as it is designed for smaller‐scale experiments. The large‐scale experiments in the text intersect with this figure at the point marked with an asterisk ~undefined), corresponding to step in and the Alternate Protocol. View Image -
Figure 17.5.3 Schematic of describing the in‐gel digestion technique and peptide extraction steps. For graphical reasons, the gel pieces are depicted as whole (to show the state of the stain), but would in practice be diced. Refer to the main body of the text for more details. Abbreviation: AMBIC, ammonium bicarbonate, pH 8.0. View Image -
Figure 17.5.4 Regeneration of GSH agarose. The expected results from are shown. Lane 1 shows the stained endogenous GST proteins present bound to a batch of GSH agarose that has been used for preclearing a tissue lysate. Lanes 2 to 4 show the sequential treatments which remove protein from the beads, and enable their reuse for preclearing procedures. View Image -
Figure 17.5.5 Sample results from a GST‐RalA pull‐down experiment. Shown is a Coomassie Blue–stained gel of a pull‐down from sheep brain cytosol using GST‐tagged RalA. For a full discussion, see Anticipated Results. Stained protein bands that have been identified by MALDI‐TOF MS are indicated on the right. Features marked with an asterisk ~undefined) are interfering species. Bands that have been identified as erroneously migrating GST‐RalA are marked G. The bands marked N are either nonspecific sheep proteins, fragments of GST‐RalA, copurified bacterial contaminants, or real nucleotide‐independent binding proteins. View Image
Videos
Literature Cited
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