A simple apparatus for generating stretched polyacrylamide gels, yielding uniform alignment of proteins and detergent micelles.
Compressed and stretched polyacrylamide hydrogels previously have been shown to offer a robust method for aligning proteins. A simple, funnel-like apparatus is described for generating uniformly stretched hydrogels. For prolate-shaped proteins, gels stretched in the direction of the magnetic field yield two-fold larger alignment than gels compressed to the same aspect ratio in this direction. Empirically, protein alignment is found to be proportional to (c-2.3)2 [(d(o/dN)3-1], where do and dN are the diameters of the cylindrical gels before and after stretching, respectively, and c is the polyacrylamide weight fraction in percent. Low gel densities, in the 4-7% range, are found to have minimal effects on macromolecular rotational correlation times, tauc, and no effect of the compression ratio on tauc could be discerned over the range studied (do/dN < or = 1.4). Application is demonstrated for a sample containing the first Ig-binding domain of protein G, and for a detergent-solubilized peptide.
Chou
J J
JJ
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Gaemers
S
S
Howder
B
B
Louis
J M
JM
Bax
A
A
D000180:Acrylic Resins / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry; D008823:Micelles; D009419:Nerve Tissue Proteins / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000295:instrumentation*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D000180:Acrylic Resins; D003902:Detergents; C029152:G-substrate; D020100:Hydrogels; D008823:Micelles; D009419:Nerve Tissue Proteins; D010455:Peptides; D011506:Proteins; C016680:polyacrylamide gels