Name: protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro
Uploaded: 2011-08-14T19:00:00
Description: Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

The work in our laboratory is supported by grants from NIH, USDA National Institute of Food and Agriculture, NSF, BARD, DOE, and BSF to V.C.

1. Expression and extraction of the proteins 
<ol>
	<li>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 larger size (e.g., GST), and the unfused tag can be used as an immobilized negative control (ProIMnc). Label the protein to be used as a soluble probe (ProSOL) with either a large or a small tag. Fuse the same tag to an appropriate negative control soluble protein (ProSOLnc) and include in the interaction assay to confirm...

<ol>
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This approach is suitable for testing protein-protein interactions between combinations of the proteins,when at least one of which the proteins is readily soluble in the binding buffer, and was successfully applied to other combination of proteins 17,18. The iInteractions between the proteins that are both insoluble under these conditions cannot be tested by this protocol. 
Also, successful refolding of ProIM is critical for the assay. Rinsing the membrane in TBS after the electro...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td>Protein assay kit</td>
 <td>Bio-Rad</td>
 <td>500-0001</td>
 </tr>
 <tr>
 <td>Proteinase inhibitor cocktail </td>
 <td>SIGMA </td>
 <td>S8820 </td>
 </tr>
 <tr>
 <td>Mini-PROTEAN system </td>
 <td>Bio-RAD </td>
 <td>165-8000 </td>
 </tr>
 <tr>
 <td>Semi-dry western blotting SD electrotransfer system </td>
 <td>Bio-RAD </td>
 <td>170-3940 </td>
 </tr>
 <tr>
 <td>Anti-rabbit IgG antibody conjugated with horse radish peroxidase </td>
 <td>GenScript </td>
 <td>A00098 </td>
 </tr>
 <tr>
 <td>Anti-GST rabbit polyclonal antibody </td>
 <td>GenScript </td>
 <td>A00097 </td>
 </tr>
 <tr>
 <td>Anti-strepII </td>
 <td>GenScript </td>
 <td>A00626 </td>
 </tr>
 <tr>
 <td>BioTrace, NT nitrocellulose transfer membrane </td>
 <td>Pall Scientific </td>
 <td>27377-000 </td>
 </tr>
 <tr>
 <td>Immobilon western chemiluminescent HRP substrate </td>
 <td>Millipore </td>
 <td>WBKL S0 050 </td>
 </tr>
 </tbody>
</table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

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&#x2019; interactions, the fluorescent signal is reconstituted3-6. 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 interactions7.
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 transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

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

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

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

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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