Expression and Purification of <i>lacZ</i> and <i>trpE</i> Fusion Proteins

Hoey, Timothy

doi:10.1002/0471142727.mb1605s28

Cited by 3 publications

(2 citation statements)

References 14 publications

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“…The plasmid was transformed to TOP10 Escherichia coli competent cells. Transformant colonies were grown in a tryptophan-deficient medium, expression of the TrpE-Repo fusion protein was induced by supplementing the medium with indoleacrylic acid (40 μg/ml), and the fusion protein was recovered in inclusion bodies [protocol adapted from ( 30 )]. Mouse immunization and serum collection were performed by Davids Biotechnologie GmbH.…”

Section: Methodsmentioning

confidence: 99%

Crustacean leg regeneration restores complex microanatomy and cell diversity

et al. 2022

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Animals can regenerate complex organs, yet this process frequently results in imprecise replicas of the original structure. In the crustacean Parhyale , embryonic and regenerating legs differ in gene expression dynamics but produce apparently similar mature structures. We examine the fidelity of Parhyale leg regeneration using complementary approaches to investigate microanatomy, sensory function, cellular composition, and cell molecular profiles. We find that regeneration precisely replicates the complex microanatomy and spatial distribution of external sensory organs and restores their sensory function. Single-nuclei sequencing shows that regenerated and uninjured legs are indistinguishable in terms of cell-type composition and transcriptional profiles. This remarkable fidelity highlights the ability of organisms to achieve identical outcomes via distinct processes.

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Section: Methodsmentioning

confidence: 99%

Crustacean leg regeneration restores complex microanatomy and cell diversity

et al. 2022

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“…Transfectants were cultured with 2-fold TY medium [6] at 37 C, and the expression of the foreign gene was induced by the addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG). After a 4 h-incubation, the cells were harvested and lysed by ultra-high pressure (700 bars) using a Mini-Lab (Rannie, Copenhagen, Denmark) at 4 C. Inclusion bodies were harvested and solubilized according to the standard procedure [7]. Fifty milligrams of proteins derived from the inclusion bodies was digested with site-specific protease Factor Xa (100 µg, Danex Biotek, Mundelstrup, Denmark) at 25 C for 4 h to separate the mouse pRb fragment from GST.…”

Section: Preparation Of Polyclonal Anti-mouse Prb Antibodiesmentioning

confidence: 99%

Sensitive Detection of Rodent Rb Protein by Polyclonal Antibodies Against a Bacterially Expressed Mouse Rb Protein.

Kobayashi

Yamaguchi

Tanaka

1998

Journal of Reproduction and Development

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Abstract. Polyclonal antibodies against the bacterially expressed mouse retinoblastoma gene product (pRb) were prepared and characterized to enable sensitive recognition of rodent pRb. The cDNA fragment corresponding to amino acids 237-595 of the mouse pRb sequence was expressed at a high level in E. coli as a fusion protein with glutathione S-transferase. Polyclonal anti-mouse pRb antibodies, MKS-1, were prepared by immunizing rabbits with the truncated pRb obtained from the fusion protein. MKS-1 sensitively recognized the hyper-and the hypophosphorylated pRbs not only in the rodent cells but also in primate cells by Western blotting following immunoprecipitation. An increase in the cellular content of total and hypophosphorylated pRbs during myogenic differentiation of mouse C2 cells was also detected quantitatively by MKS-1. Thus, MKS-1 is useful for the quantitative detection and the analysis of pRb in cell lines established from rodent tissues. Key words: Antibody, Rodent, Retinoblastoma protein, Immunoprecipitation, Western blotting.etinoblastoma gene (Rb-1) product, pRb, is known as one of the key regulators of growth, (J. Reprod. Dev. 44: [209][210][211][212][213][214] 1998) The predicted amino acid sequences of the mouse and the human pRbs are closely related (91% identity [3]), however, no antibodies that sensitively recognize the mouse pRb are available. Thus, the application of useful cell lines established from mice for analysis of the biological functions of pRb has been limited.In this presently reported study, we prepared and characterized polyclonal antibodies against a mouse pRb fragment expressed in E. coli. The antibodies sensitively detect both rodent and primate pRbs. . Several lines of evidence indicate that the phosphorylation of pRb leads to the inactivation of its growth suppressive activity [1]. As the cellular content of pRb is low, specific antibodies are required to examine the roles of this protein. To date, several monoclonal antibodies against human pRb have been obtained [4].

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Antibody response to a Babesia bigemina rhoptry-associated protein 1 surface-exposed and neutralization-sensitive epitope in immune cattle

et al. 1994

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Protective immunity against Babesia bigemina is hypothesized to involve antibodies directed against merozoite surface-exposed epitopes. Levels of antibody against a rhoptry-associated protein 1 (RAP-1) B-lymphocyte epitope, defined by surface-reactive and inhibitory monoclonal antibodies, in immune cattle sera were determined. All cattle produced antibodies to the epitope; however, there was limited correlation between immune protection induced by infection or RAP-1 immunization and the level of antibody to the neutralization-sensitive B-lymphocyte epitope examined.

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Expression and Purification of lacZ and trpE Fusion Proteins

Cited by 3 publications

References 14 publications

Crustacean leg regeneration restores complex microanatomy and cell diversity

Crustacean leg regeneration restores complex microanatomy and cell diversity

Sensitive Detection of Rodent Rb Protein by Polyclonal Antibodies Against a Bacterially Expressed Mouse Rb Protein.

Antibody response to a Babesia bigemina rhoptry-associated protein 1 surface-exposed and neutralization-sensitive epitope in immune cattle

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