Evaluation of Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Purified Proteins of Mycoplasma gallisepticum and M. synoviae as Antigens in a Dot-Enzyme-Linked Immunosorbent Assay

1990 ◽  
Vol 34 (3) ◽  
pp. 575 ◽  
Author(s):  
Alan P. Avakian ◽  
Stanley H. Kleven
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Immunosorbent Assay ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mycoplasma Gallisepticum ◽  
Polyacrylamide Gel ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dodecyl Sulfate

Using Cell-Based Activity Assays In Support Of Carryover Calculations In Multi-Product Facilities

2021 ◽  
Vol 27 (4) ◽  
Author(s):  
Gabriella del Hierro ◽  
Emily Holz ◽  
Ed Contreras ◽  
Pauline Che ◽  
Shelley Elvington ◽  
...  
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Immunosorbent Assay ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Analytical Techniques ◽  
Polyacrylamide Gel ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Cell ◽  
Therapeutic Antibodies

The calculation of cleaning carryover limits in multi-product facilities can be based on the inactivity of molecules after exposure to cleaning conditions if the inactivation of active molecules can be demonstrated. The demonstration of inactivation has been addressed in several publications that have shown degradation and/or denaturation using different analytical techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay, which directly or indirectly demonstrate that the product residue is no longer active. In this paper, authors expand the assay options by demonstrating the use of molecule-specific cell-based activity assays, which provide a “catch all” measurement of sample bioactivity, to assess the inactivation of therapeutic antibodies after exposure to cleaning conditions.

Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme

1982 ◽  
Vol 2 (8) ◽  
pp. 993-1001
Author(s):  
D Wang ◽  
Y Furuichi ◽  
A J Shatkin
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Hela Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Enzyme Complex ◽  
Cell Nuclei ◽  
Dodecyl Sulfate ◽  
Enzyme Intermediate

Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [alpha-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of approximately 68,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PPi required the presence of [alpha-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [beta-32P]GTP, [alpha-32P]CTP, [alpha-32P]UTP, or [alpha-32P]ATP did not label the approximately 68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4 degrees C were 30% or more of those obtained at 37 degrees C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PPi to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini.

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