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230804s2023 xx o 000 0 eng d |
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|a YDX
|b eng
|c YDX
|d SFB
|d OPELS
|d UKMGB
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|a GBC3B7168
|2 bnb
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|a 021096310
|2 Uk
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|a 9780443221019
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|a 0443221014
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|a (OCoLC)1392046271
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|a QP551
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|a 572.6
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|a Modifications and targeting of protein termini.
|n Part B /
|c edited by Thomas Arnesen.
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|a [S.l.] :
|b Academic Press,
|c 2023.
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|a 1 online resource.
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|a text
|2 rdacontent
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|a computer
|2 rdamedia
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|a online resource
|2 rdacarrier
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|a Methods in enzymology ;
|v v. 686
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|a Chapter Five: In vitro production of N-degron fused proteins and its application -- 1. Introduction -- 2. Key resources -- 2.1. Key resources table -- 2.2. Materials -- 2.3. Buffer recipes -- 3. Preparation of N-degron attached ZZ-domain of p62/SQSTM1 -- 3.1. Molecular cloning LC3B fused human p62 ZZ-domain -- 3.1.1. PCR amplification of ZZ-p62 (126-180) -- 3.1.2. Digestion of insert PCR product -- 3.1.3. Digestion of the pET-His-LC3B vector -- 3.1.4. Ligation -- 3.1.5. Transformation -- 3.1.6. Preparation of plasmids -- 3.1.7. Notes -- 3.2. Expression of N-degron fused ZZ-p62 -- 3.2.1. Cell culture -- 3.2.2. Cell harvest -- 3.2.3. Cell lysis -- 3.2.4. Notes -- 3.3. Purification of N-degron fused ZZ-p62 -- 3.3.1. His-affinity column chromatography -- 3.3.2. Ion exchange column chromatography -- 3.3.3. LC3B-tag cleavage and removal -- 3.3.4. Size exclusion chromatography -- 4. Preparation of human ATG4B protease -- 4.1. Purification of human ATG4B -- 4.1.1. His-affinity column chromatography -- 4.1.2. Ion exchange column chromatography -- 5. Applications of N-degron fused ZZ-p62 or N-degron fused proteins of interest -- 5.1. Crystallization of REEED-ZZ p62 -- 5.2. Notes -- 5.3. Structure determination -- 5.4. Other applications of LC3B-fusion technique -- 6. Conclusions -- Acknowledgments -- References -- Chapter Six: TEV protease cleavage in generation of artificial substrate proteins bearing neo-N-termini -- 1. Introduction -- 2. General method and equipment -- 3. Construction of a self-cleaving fusion protein -- 3.1. Materials and equipment -- 3.2. Procedure -- 3.3. Notes -- 4. Construction of fusion proteins containing the TEV protease recognition site -- 4.1. Materials and equipment -- 4.2. Procedure -- 4.3. Notes -- 5. Expression of proteins -- 5.1. Materials and equipment -- 5.2. Procedure -- 5.3. Notes -- 6. SDS PAGE and western blot.
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|a 6.1. Materials and equipment -- 6.2. Procedure -- 7. Summary and conclusion -- Acknowledgments -- References -- Chapter Seven: Affinity isolation and biochemical characterization of N-degron ligands using the N-recognin, ClpS -- 1. Introduction -- 2. Determine N-degron specificity of ClpS (N-recognin) using immobilized peptide arrays -- 2.1. Materials and buffer recipes -- 2.2. Preparation of peptide array (for first use) -- 2.3. Incubation of the N-recognin (ClpS) with the peptide array and preparation for transfer to PVDF membrane -- 2.4. Transfer of the bound N-recognin to PVDF membranes -- 2.5. Visualization and analysis of the transferred N-recognin (on PVDF membrane) -- 2.6. Regeneration, reuse and storage of peptide arrays -- 3. ClpS affinity chromatography to isolate (and identify) natural N-degron substrates/ligands -- 3.1. Materials and buffer recipes -- 3.2. Preparation and equilibration of Ni-NTA agarose beads -- 3.3. Bind ClpS-His10 (WT and mutant) to Ni-NTA agarose -- 3.4. Affinity purification of N-degron ligands from a bacterial lysate (all steps are performed at 4C) -- References -- Chapter Eight: Monitoring the interactions between N-degrons and N-recognins of the Arg/N-degron pathway -- 1. Introduction -- 2. Preparation of N-degrons for pulldown assays -- 2.1. X-peptides carrying type 1 or type 2N-degrons -- 2.1.1. Equipment -- 2.1.2. Buffers and reagents -- 2.1.3. Procedure -- 2.1.4. Notes -- 2.2. Biotin-conjugated chemical mimics of the N-degrons -- 2.2.1. Equipment -- 2.2.2. Buffers and reagents -- 2.2.3. Procedure -- 2.2.4. Notes -- 3. Preparations of N-recognins for pulldown assays -- 3.1. Extracts containing N-recognins in cultured cells -- 3.1.1. Equipment -- 3.1.2. Buffers and reagents -- 3.1.3. Procedure -- 3.1.4. Notes -- 3.2. Extracts containing N-recognins from murine tissues -- 3.2.1. Equipment.
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|a 3.2.2. Buffers and reagents -- 3.2.3. Procedure -- 3.2.4. Notes -- 3.3. Purification of N-recognins expressed in bacteria -- 3.3.1. Equipment -- 3.3.2. Buffers and reagents -- 3.3.3. Procedure -- 3.3.4. Notes -- 4. N-degron pulldown assays -- 4.1. Pulldown assays using crude lysates -- 4.1.1. Equipment -- 4.1.2. Buffers and reagents -- 4.1.3. Procedure -- 4.1.4. Notes -- 4.2. Pulldown assays with purified proteins -- 4.2.1. Equipment -- 4.2.2. Buffers and reagents -- 4.2.3. Procedure -- 4.2.4. Notes -- 4.3. Competitive inhibition of N-degron pulldown assays -- 4.3.1. Equipment -- 4.3.2. Buffers and reagents -- 4.3.3. Procedure -- 4.3.4. Notes -- 5. Monitoring the proteome captured by N-degrons -- 5.1. Immunoblotting analysis -- 5.1.1. Equipment -- 5.1.2. Buffers and reagents -- 5.1.3. Procedure -- 5.1.4. Notes -- 5.2. Liquid chromatography-mass spectrometry (LC/MS) analysis -- 5.2.1. Equipment -- 5.2.2. Buffers and reagents -- 5.2.3. Procedure -- 5.2.4. Notes -- 6. Pulldown of N-degron substrates using ubiquitin-fusion technique (UFT) -- 6.1. Equipment -- 6.2. Buffers and reagents -- 6.3. Procedure -- 6.4. Notes -- 7. Summary and conclusions -- Acknowledgments -- References -- Chapter Nine: In vitro autoubiquitination activity of E3 ubiquitin ligases of the N-degron pathway -- 1. Introduction -- 2. General method and equipment -- 3. Design of constructs -- 4. Recombinant protein expression of E3 ligase -- 4.1. Materials and equipment -- 4.2. Procedure -- 4.3. Notes -- 5. Protein purification after recombinant expression via IMAC -- 5.1. Materials and equipment -- 5.2. Procedure -- 5.3. Notes -- 6. In vitro autoubiquitination assay -- 6.1. Materials and equipment -- 6.2. Procedure -- 6.3. Notes -- 7. Western blot analyses -- 7.1. Materials and equipment -- 7.2. Procedure -- 7.3. Notes -- Acknowledgments -- References.
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|a Proteins.
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| 650 |
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|a Enzymology.
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| 776 |
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|i ebook version :
|z 9780443221019
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| 776 |
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|c Original
|z 0443221006
|z 9780443221002
|w (OCoLC)1375058080
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|u https://sciencedirect.uam.elogim.com/science/bookseries/00766879/686
|z Texto completo
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