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Application of transfer onto PVDF and nitrocellulose membranes in the purification and structural analyses of proteins

100%

Szewczyk B., Bienkowska-Szewczyk K.

Biotechnologia

1996

issue 1

129-150

Replicas of SDS-PAGE gels widen the range of application of electrophoresis to the studies of diverse biological properties of proteins as well as to the elucidation of their structures. Most often proteins are transferred to membranes made of polyvinylidene difluoride (PVDF) or nitrocelulose.Usually, a "semi-dry" blotting apparatus is used for electrophoretic transfer.SDS-PAGE gel replicas are used for the studies of these biological properties which cannot be studied directly on polyacrylamide gels. The major applications of this technique is in the immunoblotting which allows for the antigen-antibody reaction on the membrane.This reaction, after necessary amplification, is visualized with a variety of reagents.Protein microsequencing is another important application of SDS-PAGE gel replicas.In this case, a replica is stained with a general protein stain, and a band of interest is excised and used to obtain partial or complete amino acid sequence in an automatic sequencer.

Monoclonal antibodies against isoprenoid compounds

88%

Wanke M., Porebska P. A., Porebska A., Walajtys-Rode W.-R. E., Walajtys-Rode E.

Biotechnologia

2001

issue 3

169-177

Monoclonal antibodies against isoprenoid compounds were obtained by in vivo immunization of Balb/c mice with UQ-10 (ubiquinone-10) incorporated into cholesterol rich particles containing metylated bovine serum albumin (CRP-MBSA) in order to induce a strong immune response. The specificity of antibodies was evaluated by a modified dot-blot technique using Hybond-N+ membrane (Amersham). Selected antibodies III-5 (IgG3), IV-3 (IgG2b) and VII-11 (IgM) were capable of recognizing different isoprenoid compounds: ubiquinone, plastoquinone, solanesol, dolichol, -sistosterol but did not react with phospholipids, triacylglycerols or fatty acids. These antibodies may be used in immunoblotting for identifying isoprenoids (e.g. ubiquinone) in partially purified plant extracts after TLC chromatography on plastic RP-18 plates (Merck). Immunodetection of isoprenoids was performed using secondary goat anti-mice immunoglobin antibodies conjugated with biotin, extravidine-AP and BCIP/NBT as substrate

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2 Biotechnologia

1 2001

1 1996

1 Bienkowska-Szewczyk K.

1 Porebska A.

1 Porebska P. A.

1 Szewczyk B.

1 Walajtys-Rode E.

1 Walajtys-Rode W.-R. E.

1 Wanke M.

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Application of transfer onto PVDF and nitrocellulose membranes in the purification and structural analyses of proteins

Monoclonal antibodies against isoprenoid compounds