Analysis by Sodium Dodecyl Sulfate Polyacrylamide gel Electrophoresis and Western Blot of Nonspecific and Specific Viral Proteins Frequently Detected in Different Antigen Preparations of Bovine Leukemia Virus

Abstract

Abstract. Bovine leukemia virus (BLV) infection in cattle is seldom manifested clinically, and is routinely diagnosed by serologic tests such as enzyme-linked immunosorbent assay or Western blot (WB). Because of the difficulty in interpreting WB results, the aim of the present study was to determine which of the bands observed in WB were specifically produced by BLV and which corresponded to nonspecific proteins, either derived from medium components or of a cellular nature. Five different BLV antigen preparations from 2 cell lines (FLK-BLV and BLV-bat 2 ) frequently used for the production of BLV antigen were compared. The protein profiles of these antigen preparations were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and WB. Fetal calf serum, required for cellular growth and important in induction of viral transcription in vitro, was identified as a source of irrelevant proteins. In this study, 15 nonspecific protein bands in the growth medium were observed. These bands interfered with the interpretation of results. A nonspecific protein (25 kD) that was highly reactive in cell lysate preparation from BLV-bat 2 was also detected. The unequivocal identification of protein bands, both specific and nonspecific, seen in WB is important not for understanding the protein profile of antigen preparations but also for determining if an animal is BLV positive or negative.

Resistance to cerebral ischemic injury in UCP2 knockout mice: evidence for a role of UCP2 as a regulator of mitochondrial glutathione levels

“…Succinate dehydrogenase was used as an indice of mitochondrial fraction location (Bancroft 1982) and was compared with the other fractions (see below). We then proceed for gel electrophoresis and immunodetection as previously described (Llames et al . 2000).…”

“…Succinate dehydrogenase was used as an indice of mitochondrial fraction location (Bancroft 1982) and was compared with the other fractions (see below). We then proceed for gel electrophoresis and immunodetection as previously described (Llames et al 2000). Primary antibodies to Mn SOD (Calbiochem, San Diego, CA, USA, 1/200), cytochrome c antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1/200) or cytochrome oxidase (COX) subunit IV (Molecular Probes, Eugene, OR, USA) were used.…”

SDS Electrophoresis on Gradient Polyacrylamide Gels as a Semiquantitative Tool for the Evaluation of Proteinuria

“…The lack of information on the composition of the commercially available standard and the possibility of proteolytic degradation of urinary proteins when passing through urinary tracts and during sample storage may result in differences in the electrophoretic mobility of urine proteins and the components of the standard. The methodological differences in the western blot include different percentages of polyacrylamide gels, different membranes (polyvinylidene difluoride-PVDF, nitrocellulose), and antibodies conjugated with different enzymes do not always allow to detect the same proteins with the method [36][37][38]. The identification of proteins using mass spectrometry also depends on the variant of SDS electrophoresis, type of staining, method of sample preparation, and its storage [39,40].…”

Selection of peptides for serological detection of equine infectious anemia

“…Besides, EIAV cultures are time-consuming and costly for large-scale antigen production (Alvarez et al, 2007b). A number of factors must be considered in the selection of antigenic preparations, such as time spent, the amount of material needed, the complexity and purity of viral proteins, the equipment available, and the costs for obtaining the final product (Llames et al, 2000).…”