| First Author | Luo L | Year | 2014 |
| Journal | Proteomics | Volume | 14 |
| Issue | 1 | Pages | 19-23 |
| PubMed ID | 24259493 | Mgi Jnum | J:243622 |
| Mgi Id | MGI:5909201 | Doi | 10.1002/pmic.201300315 |
| Citation | Luo L, et al. (2014) Single-step protease cleavage elution for identification of protein-protein interactions from GST pull-down and mass spectrometry. Proteomics 14(1):19-23 |
| abstractText | The study of protein-protein interactions is a major theme in biological disciplines. Pull-down or affinity-precipitation assays using GST fusion proteins have become one of the most common and valuable approaches to identify novel binding partners for proteins of interest (bait). Non-specific binding of prey proteins to the beads or to GST itself, however, inevitably complicates and impedes subsequent analysis of pull-down results. A variety of measures, each with inherent advantages and limitations, can minimise the extent of the background. This technical brief details and tests a modification of established GST pull-down protocols. By specifically eluting only the bait (minus the GST tag) and the associated non-specific binding proteins with a simple, single-step protease cleavage, a cleaner platform for downstream protein identification with MS is established. We present a proof of concept for this method, as evidenced by a GST pull-down/MS case study of the small guanosine triphosphatase (GTPase) Rab31 in which: (i) sensitivity was enhanced, (ii) a reduced level of background was observed, (iii) distinguishability of non-specific contaminant proteins from genuine binders was improved and (iv) a putative new protein-protein interaction was discovered. Our protease cleavage step is readily applicable to all further affinity tag pull-downs. |
