A method to purify enzymes utilizing their specific biological affinity and catalytic specificity is described. For this chromatographic technique, an enzyme binds immobilized substrate coupled to a column in the absence of a cofactor required for catalysis but permissive for substrate binding. After washing, the missing cofactor is added to the column mobile phase, and the enzyme converts substrate into product and elutes from the column. A single-step purification of EcoRI endonuclease using a sequence-specific DNA column (containing the GAATTC motif coupled to cyanogen bromide-activated Sepharose 4B) binds EcoRI in the absence of Mg2+ and elutes when Mg2+ is applied in a highly purified state. Although the method described is specific for EcoRI, it can be readily modified for the purification of DNA polymerases and other enzymes. Furthermore, many of the same materials are also used for transcription factor purification. This protocol can be completed within 4-6 d.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1038/nprot.2006.439 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4, CD8, Caspase-3 and NF-κB in mice.
BMC Vet Res
October 2023
Department of Parasitology and Animal Diseases, Veterinary Research Institute, National Research Centre, El Buhouth Street, Dokki, Cairo, Egypt.
Background: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4, CD8, Caspase-3, and nuclear factor kappa B (NF-κB).
View Article and Find Full Text PDFAutoantibodies to beta tubulin in autoimmune liver diseases-Relation to pANCA and clinical relevance.
Clin Exp Immunol
April 2024
Department of Hematology, Oncology, Clinical Immunology and Rheumatology, University of Tuebingen, Tuebingen, Germany.
There was evidence that perinuclear antineutrophil cytoplasmic antibodies (pANCA) in autoimmune liver diseases react with human beta-tubulin-5 (TBB5). Here, we reevaluate the specificity and clinical relevance of anti-TBB5 antibodies. Patients with untreated autoimmune hepatitis (AIH; n = 53), AIH under immunosuppressive therapy (AIH-IS; n = 125), primary sclerosing cholangitis (PSC; n = 40), primary biliary cholangitis (PBC; n = 250), nonautoimmune liver diseases (n = 158), inflammatory bowel diseases (IBD; n = 30), and healthy individuals (n = 62) were tested by enzyme-linked immunosorbent assay for IgG- and IgA-antibodies against recombinant human TBB5.
View Article and Find Full Text PDFDioclea violacea lectin increases the effect of neomycin against multidrug-resistant strains and promotes the purification of the antibiotic in immobilized lectin column.
Int J Biol Macromol
May 2023
Agrarian and Biodiversity Sciences Center, Federal University of Cariri, Crato, CE, Brazil. Electronic address:
DVL is a Man/Glc-binding lectin from Dioclea violacea seeds that has the ability to interact with the antibiotic gentamicin. The present work aimed to evaluate whether the DVL has the ability to interact with neomycin via CRD and to examine the ability of this lectin to modulate the antibiotic effect of neomycin against multidrug-resistant strains (MDR). The hemagglutinating activity test revealed that neomycin inhibited the hemagglutinating activity of DVL with a minimum inhibitory concentration of 50 mM, indicating that the antibiotic interacts with DVL via the carbohydrate recognition domain (CRD).
View Article and Find Full Text PDFEvaluation of a vaccine candidate isolated from oocyst in mice.
Vet World
December 2022
Department of Parasitology and Animal Diseases, Veterinary Research Institute, National Research Centre, Dokki, Giza, Egypt.
Background And Aim: Cryptosporidiosis is a leading cause of diarrheal disease worldwide and is an animal and public health burden. This study aimed to evaluate the protective potential of affinity-purified oocyst antigen as a vaccine candidate according to fecal oocyst shedding, humoral and cellular immune responses, histopathological changes, and the number of parasite developmental stages in ileal and hepatic tissues.
Materials And Methods: We isolated oocysts from naturally infected buffalo calves and identified them molecularly as isolates (GenBank: ON730707 and ON730708) by targeting the oocyst wall protein gene.
Characterization of a novel O-acetyl sialic acid specific lectin from the hemolymph of the marine crab, Atergatis integerrimus (Lamarck, 1818).
Fish Shellfish Immunol
November 2020
Department of Zoology, Holy Cross College (Autonomous), Nagercoil, Affiliated to Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli, 627012, Tamil Nadu, India.
An O-acetyl sialic acid specific lectin was purified from the hemolymph of the marine crab Atergatis integerrimus by affinity chromatography using BSM (Bovine Submaxillary Mucin) coupled to cyanogen bromide activated Sepharose 4B and biospecific adsorption using formalinized buffalo erythrocytes. The purified AiL (Atergatis integerrimus lectin) showed an 1218 fold increase in specific activity when compared to the crude hemolymph agglutinin. The lectin, on non - denaturing PAGE showed a single band of 216 kDa and when subjected to SDS - PAGE, the lectin resolved into three subunits of molecular weight 70, 72 and 74 kDa.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!