General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays

Thomas M Tomasiak; Bjørn P Pedersen; Sarika Chaudhary; Andrew Rodriguez; Yaneth Robles Colmanares; Zygy Roe-Zurz; Sobha Thamminana; Meseret Tessema; Robert M Stroud

doi:10.1002/0471140864.ps2911s77

General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays

Thomas M Tomasiak
, Bjørn P Pedersen
, Sarika Chaudhary
, Andrew Rodriguez
, Yaneth Robles Colmanares
, Zygy Roe-Zurz
, Sobha Thamminana
, Meseret Tessema
, Robert M Stroud

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

12 Citations (Scopus)

Abstract

This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable. Curr. Protoc. Protein Sci. 77:29.11.1-29.11.14 © 2014 by John Wiley & Sons, Inc.

Original language	English
Journal	Current Protocols in Protein Science
Volume	77
Pages (from-to)	29.11.1-29.11.14
ISSN	1934-3655
DOIs	https://doi.org/10.1002/0471140864.ps2911s77
Publication status	Published - 2014
Externally published	Yes

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Tomasiak, T. M., Pedersen, B. P., Chaudhary, S., Rodriguez, A., Colmanares, Y. R., Roe-Zurz, Z., Thamminana, S., Tessema, M., & Stroud, R. M. (2014). General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays. Current Protocols in Protein Science, 77, 29.11.1-29.11.14. https://doi.org/10.1002/0471140864.ps2911s77

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title = "General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays",

abstract = "This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable. Curr. Protoc. Protein Sci. 77:29.11.1-29.11.14 {\textcopyright} 2014 by John Wiley \& Sons, Inc.",

author = "Tomasiak, \{Thomas M\} and Pedersen, \{Bj{\o}rn P\} and Sarika Chaudhary and Andrew Rodriguez and Colmanares, \{Yaneth Robles\} and Zygy Roe-Zurz and Sobha Thamminana and Meseret Tessema and Stroud, \{Robert M\}",

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year = "2014",

doi = "10.1002/0471140864.ps2911s77",

language = "English",

volume = "77",

pages = "29.11.1--29.11.14",

journal = "Current Protocols in Protein Science",

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Tomasiak, TM, Pedersen, BP, Chaudhary, S, Rodriguez, A, Colmanares, YR, Roe-Zurz, Z, Thamminana, S, Tessema, M & Stroud, RM 2014, 'General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays', Current Protocols in Protein Science, vol. 77, pp. 29.11.1-29.11.14. https://doi.org/10.1002/0471140864.ps2911s77

General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays. / Tomasiak, Thomas M; Pedersen, Bjørn P; Chaudhary, Sarika et al.
In: Current Protocols in Protein Science, Vol. 77, 2014, p. 29.11.1-29.11.14.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays

AU - Tomasiak, Thomas M

AU - Pedersen, Bjørn P

AU - Chaudhary, Sarika

AU - Rodriguez, Andrew

AU - Colmanares, Yaneth Robles

AU - Roe-Zurz, Zygy

AU - Thamminana, Sobha

AU - Tessema, Meseret

AU - Stroud, Robert M

PY - 2014

Y1 - 2014

N2 - This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable. Curr. Protoc. Protein Sci. 77:29.11.1-29.11.14 © 2014 by John Wiley & Sons, Inc.

AB - This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable. Curr. Protoc. Protein Sci. 77:29.11.1-29.11.14 © 2014 by John Wiley & Sons, Inc.

U2 - 10.1002/0471140864.ps2911s77

DO - 10.1002/0471140864.ps2911s77

M3 - Journal article

C2 - 25081745

SN - 1934-3655

VL - 77

SP - 29.11.1-29.11.14

JO - Current Protocols in Protein Science

JF - Current Protocols in Protein Science

ER -

General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays

Abstract

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