Improved separation of the common transferrin variants in gels containing pH 5–7 Ampholines and HEPES

Abstract

Improved separation of the common transferrin variants in gels containing pH 5-7 Ampholines and HEPESAn ultrathin-layer polyacrylamide gel isoelectric focusing technique is described for obtaining highly resolved transferrin (Tf) subtypes. N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer was used to flatten the pH gradient in gels containing pH 5-7 Ampholines. The result was increased separation distances for the TfC subtype bands. The method is effective even with the most recent lots of pH 5-7 Ampholines which were not capable of providing an appropriate environment for effective TfC subtyping.Subtyping of transferrin (Tf) derived from serum or bloodstains can be performed effectively by isoelectric focusing in gels containing pH 5-7 Ampholines (LKB) [l-81. Although the methodology is reliable, one constraint on the reproducibility of this approach which is outside the control of the laboratory is the lot-to-lot variation of the pH 5-7 Ampholines. Budowle [9,101 reported that lots 48 and 50 produced the desired TfC patterns demonstrated in the literature; however, it was impossible to resolve the C 1C3 phenotype when lot 49 was employed. The more recent lots 51-53 also have been found to be unsuitable for resolving the C 1 and C3 bands (unpublished data). This technical note describes an approach that will alleviate this problem as well as separate further the TfC subtype bands.Serum samples were obtained from donors at the FBI Academy. Rare Tf variants were kindly provided by Dale D. Dykes, Memorial Blood Center of Minneapolis, Minneapolis, Minnesota. Bloodstains were prepared as previously described [ 1 11. Ten pL of serum were mixed with 40 pL of 0.5 % wlv ferrous ammonium sulfate and incubated for 1 h at room temperature prior to isoelectric focusing [61. Cuttings (2 x 5 mm) of the bloodstains were extracted for 30 min in 20 pL of 0.5 % wlv ferrous ammonium sulfate with continuous agitation at room temperature [71. The treated serum samples and bloodstainextracts were absorbedinto Whatman#l tabs(5 x 5 mm) and LKB applicator tabs (5 x 5 mm), respectively, lightly blotted, and applied to the gel 3.5 cm from the cathode. Ultrathin-layer polyacrylamide gels (145 x 110 x 0.2 mm) were cast onto silanized glass plates [12, 131 using the flap technique [12, 131 or a Bio-Rad ultrathin-layer casting tray. The gels were allowed to polymerize at least 3 h. All gels (5 % T, 3 % C) contained 4 % w/v pH 5-7 Ampholines (LKB). Lots 43 and 46-53 of pH 5-7 Ampholines were used. Gels also were cast containing 2.35 % w/v ofthe separator N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (Sigma). The anolyte and catholyte were 0.05 M H3P04 and 0.20 MNaOH, respectively. The interelectrode wick distance was 9.5 cm. The gels were focused on an Ultrophor (LKB) using conditions previously described [61. Following ultrathinlayer polyacrylamide gel isoelectric focusing, the gels were fix-ed in 12.5 % w/v trichloroacetic acid for 20 min. The gels were either stained with Coomassie Brilliant Blue R-250 or silve...

Red cell enzymes and plasma protein polymorphisms in the population of Cabo Verde (West Africa)

“…After electrophoresis, gels were stained with amido black. Subtypes of TF C were determined by isoelectric focusing in polyacrylamide gels according to the method of Budowle (1987), with subsequent protein staining using Coomassie brilliant blue R…”