Affinity Chromatography of Bovine Pancratic Ribonuclease A

Wilchek, Meir; Gorecki, Marian

doi:10.1111/j.1432-1033.1969.tb00799.x

Cited by 105 publications

(25 citation statements)

References 18 publications

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“…Equipment that could not be autoclaved was treated with diethylpyrocarbonate (Sigma) (11). Purified IgG antibody was depleted of RNase activity by repeated passage over 5'-(4-aminophenylphosporyl)uridine 2'-(3')-phosphateagarose (Miles) columns (12). Depletion of RNase activity was assessed by incubating aliquots of the purified antibody with RNA and then measuring RNA degradation by agarose gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Milhausen¹,

Gill²,

Parker³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems.

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Section: Methodsmentioning

confidence: 99%

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Milhausen¹,

Gill²,

Parker³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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“…Similarly, as an alternative strategy to eliminate nucleases from the collagen, we purified collagen using a Sepharose column. 41,42 E-AB sensors coated with collagen I hydrogel purified by affinity chromatography did not withstand employment in blood serum (Figure 6). In summary, only collagen films prepared with RIs provided stable, long-term function in undiluted serum.…”

Section: Resultsmentioning

confidence: 99%

Collagen Membranes with Ribonuclease Inhibitors for Long-Term Stability of Electrochemical Aptamer-Based Sensors Employing RNA

Santos-Cancel

White

2017

Anal. Chem.

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Electrochemical aptamer-based (E-AB) sensors offer advantageous analytical detection abilities on account of their rapid response time (sec to min), specificity to a target, and selectivity to function in complex media. Ribonucleic acid (RNA) aptamers employed in this class of sensor offer favorable binding characteristics resulting from the ability of RNA to form stable tertiary folds aided by long-range intermolecular interactions. As a result, RNA aptamers can fold into more complex three-dimensional structures than their DNA counterparts, and consequently, exhibit better binding ability to target analytes. Unfortunately, RNA aptamers are susceptible to degradation by nucleases, and for this reason, RNA-based sensors are scarce or require significant sample pretreatment before use in clinically-relevant media. Here, we combine the usefulness of a collagen I hydrogel membrane with entrapped ribonuclease inhibitors (RI) to protect small molecule RNA E-AB sensors from endogenous nucleases in complex media. More specifically, the biocompatibility of the naturally polymerized hydrogel with encapsulated RI promotes the protection of an aminoglycoside-binding RNA E-AB sensor up to 6 hours; enabling full sensor function in nuclease-rich environments (undiluted serum) without the need for prior sample preparation or oligonucleotide modification. The use of collagen as a biocompatible membrane represents a general approach to compatibly interface E-AB sensors with complex biological samples.

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“…We have recently described the selective purification of certain enzymes [3,9], and other biological macromolecules by affinity chromatography [lo] . The protein to be purified is passed through a column containing a cross-linked polymer or gel to which a specific competitive inhibitor or ligand of the protein has been covalently attached.…”

Section: Discussionmentioning

confidence: 99%

Affinity Chromatography of Bovine Pancratic Ribonuclease A

Cited by 105 publications

References 18 publications

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Collagen Membranes with Ribonuclease Inhibitors for Long-Term Stability of Electrochemical Aptamer-Based Sensors Employing RNA

The use of affinity chromatography for the purification of affinity labeled peptides from staphylococcal nuclease

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