An agarose–acrylamide composite native gel system suitable for separating ultra-large protein complexes
Section snippets
Materials
Acrylamide stock solution (30% acrylamide, 37:1 cross-linking ratio) was purchased from National Diagnostics (GA, USA). TEMED was obtained from EM Biosciences. SeaKem Gold agarose (Cambrex) was used unless mentioned otherwise. All other chemical reagents were purchased from Sigma (MO, USA). Protean II xi gel combs (20-well with 0.5 mm thickness) were purchased from Bio-Rad. The High-Molecular-Weight Calibration Kit for native electrophoresis containing thyroglobulin, ferritin, catalase, and
A triple-comb technique for forming sample wells in agarose–acrylamide CNG
Even though agarose was added to strengthen the acrylamide gel, we found that ordinary single-pieced combs were unsuitable for forming sample wells, due to the fragility and the gluey nature of the composite gel. Usually, walls of the wells remained stuck on the comb, making it difficult to remove the comb without breaking the walls. Tatsumi and Hattori [9] solved this problem by introducing air into the sample wells with thin needles inserted between the walls and the comb. This technique
Discussion
Native gel electrophoresis is a valuable method for identifying and analyzing multiprotein assembles, and it has the potential to be utilized more widely to address macromolecular complexes of excessive molecular masses. The usefulness of native electrophoretic analysis is well demonstrated by the blue native polyacrylamide gel electrophoresis (BN-PAGE), a modified version of the technique developed to suit the separation of membrane proteins [15], [16] such as ATP synthase. Applications of
Acknowledgments
We thank Drs. Stewart Shuman (Sloan-Kettering Institute), Averell Gnatt (U. Maryland Schl. Med.), and Jeffrey Roberts (Cornell U.) for sharing reagents. Work presented here has been supported in part by a grant from NIH (GM064651) to J.F. and an institutional grant from American Cancer Society (IRG-01-231-01).
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