Electroblotting proteolytic products from native gel for direct N-terminal sequence analysis: an approach for studying protein-protein interaction.
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.
Wang
F
F
Biotechnology Department, Hoffmann-La Roche Inc., Nutley, NJ 07110.
Su
C
C
Hollfelder
K
K
Waddington
D
D
Pan
Y C
YC
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli; D007376:Interleukin-2 / Q000737:chemistry* / Q000378:metabolism; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry*; D015375:Receptors, Interleukin-2 / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D005465:Fluorocarbon Polymers
D019286:Endopeptidase K; D010450:Endopeptidases; D007376:Interleukin-2; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014357:Trypsin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers