Electroblotting proteolytic products from native gel for direct N‐terminal sequence analysis: An approach for studying protein‐protein interaction

Abstract

Proteins which are electroblotted from native gels onto polyvinylidene difluoride (PVDF) membranes are suitable for detailed structural analysis. This method, in conjunction with limited proteolysis and N-terminal sequencing, has been used to study the molecular interactions between native protein molecules. The interaction between recombinant interleukin-2 (rIL-2) and its receptor (rIL-2R alpha) was examined as a model system. The working strategy consists of (i) proteolysis of rIL-2R alpha and rIL-2R alpha/rIL-2 complex, (ii) separation of the major proteolytic products by native polyacrylamide gel electrophoresis followed by electroblotting onto PVDF membrane, and (iii) sequence analysis of the blotted protein bands for the identification of peptide regions sensitive to proteolysis. Results have indicated that the exon 3 encoded region in rIL-2R alpha is sensitive to proteolysis regardless whether it is complexed with rIL-2 or not. This suggests that no major conformational changes occur in rIL-2R alpha during interaction with rIL-2. This electroblotting approach is, therefore, useful for studying protein-protein interaction in solution.

Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor

“…Native gel electrophoresis proved to be a simple and effective technique in this instance, especially for the isolation of the complex and determination of its stoichiometry. The mobility shift assay, successfully employed in the detection of other protein-protein complexes [35][36][37][38], is primarily suited to studying complexes of high affinity, such as the one between deoxyhypusine synthase and ec-eIF5A. It has several advantages over conventional gel filtration, which suffers from low resolution, especially when the difference in molecular mass between the complex and a component protein is small, as is the case for the complex (181 kDa) of deoxyhypusine synthase (164 kDa) with ec-eIF5A (" 17 kDa).…”

Refinement of the Differentiated Phenotype of the Spermatogenic Cell Line GC-2spd(ts)'1

“…One part extract was added to one part double-strength Laemmli buffer containing 2% -mercaptoethanol [23], and the samples were heated for 5 min at 90°C. Proteins were separated on a 10% separating and 5% stacking polyacrylamide gel under nonreducing nondenaturing (native) electrophoretic conditions [38] and electrotransferred according to the method of Towbin et al [39]. The nitrocellulose was blocked for 30 min with 5% milk in PBS, washed with a PBS/0.5% Tween-20 solution, and incubated with monoclonal antibody ascites overnight at 5C at the following concentrations: MHS-10 (1:5000), HS-63 (1:100), and null ascites (1:100).…”

Proteomics and Non-proteomics Approaches to Study Stable and Transient Protein-Protein Interactions

“…It is known, for example, that gel electrophoresis is the low-throughput method and that the gel electrophoresis-based methods are difficult to automate. BN-PAGE has low resolution [122], and CN-PAGE has even a lower resolution than BN-PAGE [155,156]. As an alternative to BN-PAGE and CN-PAGE, capillary electrophoresis (CE) was suggested as a rapid, highly sensitive, quantitative, high-resolution method for investigation of PPI [157].…”