Technical problems concerning the use of immunoblots for the detection of antinuclear antibodies.
When the immunoblotting technique is used as a diagnostic tool, the reproducibility of the results is a major problem. When purified radiolabelled proteins were applied onto SDS gels, the recovery of radioactivity on the blot after electrophoresis, blotting and incubation ranged from 10 to 65%, depending on the protein. Although the addition of SDS was subsequently shown to improve protein transfer from gel to blot, it is not recommended because immunological recognition of proteins is diminished after this transfer step. We suggest that during the incubation of protein blots detergents are necessary not only to diminish non-specific background, but also to renature proteins. However, since these detergents also elute protein from nitrocellulose and other blotting matrices, they are in part responsible for the lack of reproducibility in immunoblotting results.
Van Dam
A P
AP
Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam, The Netherlands.
Van den Brink
H G
HG
Smeenk
R J
RJ
D000974:Antibodies, Antinuclear / Q000032:analysis*; D001323:Autoantibodies / Q000032:analysis / Q000276:immunology; D002021:Buffers; D003101:Collodion; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005174:Factor XII / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis; D011485:Protein Binding; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000032:analysis; D012967:Sodium Dodecyl Sulfate
D000974:Antibodies, Antinuclear; D001323:Autoantibodies; D002021:Buffers; D003902:Detergents; D007074:Immunoglobulin G; D012709:Serum Albumin; D012967:Sodium Dodecyl Sulfate; D005174:Factor XII; D003101:Collodion