Pseudo‐ligand affinity chromatography and high‐voltage isoelectric focusing as a multiparameter method for separation and identification of plasma proteins

A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from fresh human bloodstains by ultrathin-layer polyacrylamide gel isoelectric focusing. Electrofocusing of phosphoglucomutase on 0.2 mm thick gels took 52 and 80 min on the Cold Focus Apparatus and Ultrophor, respectively, when the distance separating the electrodes was 8.0 cm, whereas a 9.5 cm distance between electrode wicks required 95 min on the Multiphor to complete isoelectric focusing. The gels with 8.0 cm between electrode wicks provided sharper band patterns compared with the gels with 9.5 cm between electrode wicks.

Phosphoglucomutase‐1 subtyping of human bloodstains on ultrathin‐layer polyacrylamide gels

Budowle¹

1984

Typing of esterase D by isoelectric focusing

Budowle

1984

staining was negligible when using peroxidase-labelled probes, in contrast to the 1251-labelled antisera where the increased background staining reduced the sensitivity.The use of glutaraldehyde vapor fixation of the immobilized antigens followed by reduction with 0.02 % NaBH4 before performing the immunoassays (71 did not significantly improve the sensitivity, although slightly enhanced staining was observed.The conditions used in these immunoassays may differ from those used by other investigators and may be more or less sensitive compared to others. Therefore, the detection limits do not represent ultimate values, but serve as method to compare the various immunoassays. We conclude that the double sandwich immunoassay is the most attractive approach to detect antibody bound to nitrocellulose immobilized antigen since a) it is most sensitive technique and b), it reduces the number of incubation steps compared to other methods.

Transferrin subtypes determined by ultrathin‐layer polyacrylamide gel isoelectric focusing

Budowle

1985

Electrophoresis 1985.6. 97-99 Transferrin subtyping 97 results in premature cooling of the frame and thus premature gelling. Also, bubble formation is a problem. Further, the gel is often damaged when removed from the frame. Another means of open casting used in some laboratories is an IR lamp and glass plates. We elected not to use IR in our laboratory since IR radiation is injurious to the lens of the eye and because the temperature of the heated surface is difficult or impossible to control. The heating-cooling plate described here eliminates all of the undesirable pitfalls of closed casting as well as those of open casting with an IR source. Obviously, it is imperative that the surface upon which the agarose is poured be perfectly level to insure uniformity in the thickness ofthe gel. It is usually recommended that open casting not be used for agarose gels less than 1.5 mm in thickness (FMC Corporation, Rockland, ME), i. e., closed casting is recommended. The results presented here would appear to obviate the above recommendation. Transferrin subtypes determined by ultrathin-layer pol yacrylamide gel iS0eleCtriC focusing An ultrathin-layer polyacrylamide gel isoelectric focusing method is described for subtyping transferrin. After a one-hour pretreatment at room temperature of transferrin in serum with ferrous ammonium sulfate, the samples were subjected to isoelectric focusing for 140 min with a final voltage of 3500 V. Thus, transferrin subtyping was achieved in one-half of a working day. Population data on transferrin in Black and White samples from Baltimore, Maryland, were obtained using this method. The data were similar to that previously reported on these races from other geographical locations.Smithies, 11 using starch-gel electrophoresis, was the first to observe inherited transferrin (TO variants. The common Tf phenotype was designeted TfC, the more anodal variants were termed TfB, and the more cathodal, TfD. The allelic frequency of TfC is greater than 98 % for most populations 121. However, Kuhnl and Spielmann [3,41, using isoelectric focusing (IEF), showed that the greatest degree of heterozygosity for Tf lies in the commonly occurring TfC type. Three common subtypes of the TfC allele have been resolved by IEF: TfC 1, TfC2 and TfC3. Combinations of the 3 TfC alleles may result in one of six phenotypes: TfC 1, TfC 1-C2, TfC 1-C3, TfC2, TfC2-C3 and TfC3. Ultrathin-layer polyacrylamide gel isoelectric focusing (ULPAGIF) has been shown to be more effective than I E F for protein separation on thin-layer gels I5,61. Some of the advantages of ULPAGIF over I E F on thin-layer gels are increased resolution and sensitivity, shorter separation times, and reduced cost. The only report known to this author which used the ULPAGIF methodology for Tf subtyping I71 required 3.5 h for protein separation -an exCorrespondence: Dr. Bruce Budowle, Forensic Science Research Group, Laboratory Division, FBI Academy, Quantico, VA 22135, USA Abbreviations: Tf, Transferrin; IEF, isoelectric focusing; ULPAGIF, ultrathi...