An Improved Method for the Microscale Preparation and Characterization of Hapten−Protein Conjugates:  The Use of Cholesterol as a Model for Nonchromophore Hydroxylated Haptens

1999 ◽  
Vol 10 (6) ◽  
pp. 1143-1149 ◽  
Author(s):  
Jérôme Naar ◽  
Philippe Branaa ◽  
Mireille Chinain ◽  
Serge Pauillac
Keyword(s):  
Improved Method ◽  
Protein Conjugates

PRODUCTION AND CHARACTERIZATION OF ANTIBODIES ELICITED BY DIFFERENT ALDOSTERONE-PROTEIN CONJUGATES

1972 ◽  
Vol 68 (3_Suppl) ◽  
pp. S32
Author(s):  
W. Vetter ◽  
E. Freedlender ◽  
E. Haber
Keyword(s):  
Protein Conjugates

scholarly journals Characterization of Hapten-Protein Conjugates: Antibody Generation and Immunoassay Development for Chlorophenoxyacetic Acid Pesticides

2009 ◽  
Vol 92 (6) ◽  
pp. 1773-1779 ◽  
Author(s):  
Robin C Boro ◽  
K Vikas Singh ◽  
C Raman Suri
Keyword(s):  
Inhibitory Concentration ◽  
Dichlorophenoxyacetic Acid ◽  
Protein Conjugation ◽  
Protein Conjugates ◽  
Highly Sensitive ◽  
Ic50 Values ◽  
Chlorophenoxyacetic Acid ◽  
2,4 Dichlorophenoxyacetic Acid

Abstract The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1500 ng/mL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.

scholarly journals An Improved Method for Optical Characterization of Mineral Dust and Soot Particles in the El Paso-Juárez Airshed

Atmosphere ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 866
Author(s):  
Javier Polanco ◽  
Manuel Ramos ◽  
Rosa M. Fitzgerald ◽  
William R. Stockwell
Keyword(s):  
Mineral Dust ◽  
El Paso ◽  
Improved Method ◽  
Likelihood Estimator ◽  
Time Resolved ◽  
Soot Particles ◽  
Scattering Coefficients ◽  
Measuring Devices ◽  
Good Agreement

Highly time-resolved aerosol measurements and analysis are necessary for a proper aerosol characterization in many polluted regions, because aerosol concentrations in polluted environments can change over time scales of minutes. However, many urban measuring sites have measuring devices that provide time resolved average aerosol concentrations over a day or two at best. Light-scattering properties of mineral dust and soot particles in the El Paso-Juárez Airshed were analyzed with an improved methodology, using the T-matrix, a maximum likelihood estimator (MLE), and data from both an acoustic extinctiometer and a laser particle counter. The hourly inter-comparisons of the scattering coefficients’ results between the model and those obtained using the instruments at a wavelength of 0.87 μm show good agreement. This methodology has been applied in the El Paso-Juárez Airshed successfully, and it could be used in other cities where mineral dust and soot are major components of the aerosol concentrations.

scholarly journals New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle

1997 ◽  
Vol 325 (2) ◽  
pp. 325-330 ◽  
Author(s):  
Sung Hee BAEK ◽  
Seung Kyoon WOO ◽  
Jae LEE ◽  
Yung Joon YOO ◽  
Choong Myung CHO ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Ubiquitinated Protein ◽  
Protein Conjugates ◽  
Isopeptide Bonds ◽  
Linear Poly ◽  
Single Polypeptide ◽  
Free Ubiquitin

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). Here we report the purification and characterization of one of the UCHs, called UCH-8, with 125I-labelled ubiquitin-α-NH-MHISPPEPESEEEEEHYC as a substrate. The purified UCH-8 behaved as a 240 kDa protein on a Superdex-200 column under non-denaturing conditions but as a 130 kDa polypeptide on analysis by PAGE under denaturing conditions, suggesting that the enzyme consists of two identical subunits. Thus this enzyme seems to be distinct in its dimeric nature from other purified UCHs that consist of a single polypeptide, except that UCH-6 is also a homodimer of 27 kDa subunits. UCH-8 was maximally active between pH 7.5 and 8, but showed little or no activity below pH 7 and above pH 9. Like other UCHs it was sensitive to inhibition by thiol-blocking agents such as N-ethylmaleimide, and by ubiquitin aldehyde. The purified UCH-8 hydrolysed not only ubiquitin-α-NH-protein extensions, including ubiquitin-α-NH-carboxy extension protein of 80 amino acid residues and ubiquitin-α-NH-dihydrofolate reductase, but also branched poly-ubiquitin that are ligated to proteins through ϵ-NH-isopeptide bonds. However, it showed little or no activity against poly-His-tagged di-ubiquitin, suggesting that UCH-8 is not involved in the generation of free ubiquitin from the linear poly-ubiquitin precursors. These results suggest that UCH-8 might have an important role in the production of free ubiquitin and ribosomal proteins from their conjugates as well as in the recycling of ubiquitin molecules after the degradation of poly-ubiquitinated protein conjugates by the 26 S proteasome.

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