TY - GEN A1 - Völzke, Jule L. A1 - Hodjat Shamami, P. A1 - Gawlitza, Kornelia A1 - Feldmann, Ines A1 - Zimathies, Annett A1 - Weller, Michael G. T1 - High-purity corundum as support for affinity extractions from complex samples T2 - Preprints N2 - Nonporous corundum powder, known as an abrasive material in the industry, was functionalized covalently with protein binders to isolate and enrich specific proteins from complex matrices. The materials based on corundum were characterized by TEM, ESEM, BET, DLS, and zeta potential measurements. The strong Al-O-P bonds between the corundum surface and amino phosphonic acids are used to introduce functional groups for further conjugations. The common cross-linker glutaraldehyde was compared with a hyperbranched polyglycerol (PG) of around 10 kDa. The latter is oxidized with periodate to generate aldehyde groups that can covalently react with the amines of the surface and the amino groups from the protein via a reductive amination process. The amount of bound protein was quantified via aromatic amino acid analysis (AAAA). This work shows that oxidized polyglycerol can be used as an alternative to glutaraldehyde. With polyglycerol, more of the model protein bovine serum albumin (BSA) could be attached to the surface under the same conditions, and lower nonspecific binding (NSB) was observed. As a proof of concept, IgG was extracted with protein A from crude human plasma. The purity of the product was examined by SDS-PAGE. A binding capacity of 1.8 mg IgG per g of corundum powder was achieved. The advantages of corundum are the very low price, extremely high physical and chemical stability, pressure resistance, favorable binding kinetics, and flexible application. KW - Protein KW - Bioseparation KW - Purification KW - Immunoprecipitation KW - Affinity chromatography KW - Polyglycerol KW - Glutaraldehyde KW - Linker KW - Bioconjugation KW - Self-assembled monolayer (SAM) KW - Periodate oxidation KW - Reductive amination KW - Antibodies KW - Igg KW - Immunoglobulins KW - Carrier KW - Solid phase KW - Hyperbranched polymer KW - Aromatic amino acid analysis aaaa PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-555142 DO - https://doi.org/10.20944/preprints202208.0004.v1 SP - 1 EP - 13 PB - MDPI CY - Basel AN - OPUS4-55514 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schroeder, Barbara A1 - Le Xuan, Hoa A1 - Völzke, Jule L. A1 - Weller, Michael G. T1 - Preactivation Crosslinking - An Efficient Method for the Oriented Immobilization of Antibodies JF - Methods and Protocols N2 - Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications. KW - Antibody coating KW - Proximity-enhanced reaction KW - Immunoglobulins KW - IgG KW - Protein A KW - Protein G KW - Immunoprecipitation KW - Immunocapture KW - Regeneration KW - Biosensor KW - Immunosensor KW - Affinity chromatography KW - Immunoaffinity extraction KW - Oriented immobilization KW - Immunoassay KW - Bioconjugation PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-479786 UR - https://www.mdpi.com/2409-9279/2/2/35 DO - https://doi.org/10.3390/mps2020035 SN - 2409-9279 VL - 2 IS - 2 SP - 1 EP - 14 PB - MDPI CY - Basel, Switzerland AN - OPUS4-47978 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Schroeder, Barbara A1 - Le Xuan, Hoa A1 - Völzke, Jule L. A1 - Weller, Michael G. T1 - Preactivation crosslinking – An efficient method for the oriented immobilization of antibodies T2 - Preprints N2 - Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications. KW - Antibody coating KW - Proximity-enhanced reaction KW - Immunoglobulins KW - IgG KW - Protein G KW - Protein A KW - Immunoprecipitation KW - Immunocapture KW - Stabilization KW - Biosensor KW - Biochip KW - Microarray KW - ELISA KW - Immunoassay KW - Immunosensor KW - Crosslinker KW - Nanoparticles KW - Click chemistry KW - Herceptin KW - Trastuzumab PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-478797 DO - https://doi.org/10.20944/preprints201904.0205.v1 SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-47879 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -