Enhanced field strength and resolution in gel electrophoresis upon substitution of buffer by histidine at its isoelectric point.
Gel electrophoresis in isoelectric buffers, recently introduced by R. Westermeier and H. Schickle (Electrophoresis '95, Paris, Abstract No.3, 1995), was applied to the automated HPGE-1000 apparatus in the expectation to be able to increase the field strength under the limiting conditions of heat dissipation capacity and voltage of that apparatus. A previous attempt to achieve that aim by reduction of gel thickness had not yielded more than a twofold increment in resolving power. Replacing 0.2 X Tris-boric acid-EDTA (TBE) buffer, conventionally applied in the apparatus at 15 V/cm, by 0.05 M histidine, pH 7.6 (close to the pI of 7.47), allows one to increase the field strength to 60 V/cm, thus providing a nearly fivefold increment in resolution under otherwise identical conditions (fluorescein carboxylate-labeled conalbumin-sodium dodecyl sulfate (SDS) and soybean trypsin inhibitor-SDS samples, 10 degrees C, 4% MetaPhor agarose). An additional decrease in band dispersion can be obtained by decreasing the starting zone width through buffer dilution in the sample phase.
Chen
N
N
Section on Macromolecular Analysis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA.
Chrambach
A
A
D001888:Boric Acids / Q000737:chemistry; D002021:Buffers; D003207:Conalbumin / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004553:Electric Conductivity; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006639:Histidine; D007526:Isoelectric Point; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014325:Tromethamine / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry
D001888:Boric Acids; D002021:Buffers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D014361:Trypsin Inhibitors; C097499:fluorescein carboxylate; D014325:Tromethamine; D003207:Conalbumin; D012967:Sodium Dodecyl Sulfate; D006639:Histidine; D004492:Edetic Acid; C032688:boric acid