Improved electrophoresis and transfer of picogram amounts of protein with hemoglobin.
During electrophoresis and electroblotting to transfer membranes, picogram amounts of protein can react irreversibly with the polyacrylamide matrix, preventing complete electrophoresis and efficient electroblotting. Bovine hemoglobin, but not other potential carrier proteins, mitigates this protein loss by migrating with or ahead of other proteins and scavenging reactive groups. Inclusion of 5 micrograms of hemoglobin in sample wells increases by 4-fold the amount of a radiolabeled test protein, myosin I beta, found at its appropriate 120-kDa position in sodium dodecyl sulfate-polyacrylamide gels. For electroblotting, incubating the gel with 0.25 mg/ml hemoglobin prior to transfer improves mobilization of picogram amounts of radiolabeled myosin I beta out of the gel by about 6-fold. For picogram amounts of proteins, therefore, approximately 20-fold more protein transfers to a blotting membrane when hemoglobin is used during both electrophoresis and transfer. This effect is general: transfer of radiolabeled Drosophila embryo proteins is improved dramatically by including hemoglobin in the pretransfer incubation solution. We suggest that electroblot-based detection of small amounts of protein, particularly when in the absence of other potential carrier proteins, can be improved substantially by using hemoglobin.
Gillespie
P G
PG
Department of Physiology, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Gillespie
S K
SK
D000818:Animals; D002417:Cattle; D004330:Drosophila / Q000737:chemistry / Q000196:embryology; D004527:Egg Proteins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry*; D009218:Myosins / Q000032:analysis; D011506:Proteins / Q000032:analysis*
D004527:Egg Proteins; D006454:Hemoglobins; D011506:Proteins; D009218:Myosins