Elsevier

Analytical Biochemistry

Volume 343, Issue 1, 1 August 2005, Pages 166-175
Analytical Biochemistry

An agarose–acrylamide composite native gel system suitable for separating ultra-large protein complexes

https://doi.org/10.1016/j.ab.2005.05.016Get rights and content

Abstract

An agarose–acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500 kDa) and for analyzing protein–protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate–polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein–protein interactions as exemplified here by the formation and demonstration of RNA polymerase II–Fcp1 complex.

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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