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    TMR labeling of LRRK proteins

    Mariusz Matyszewski, David Snead

    Published: 2022-05-24 DOI: 10.17504/protocols.io.ewov1nq5ogr2/v1
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    Abstract

    Protocol for non-specific TMR labeling of LRRK1 and LRRK2 RCKW proteins.

    Protocol developed by David Snead and adapted by Mariusz Matyszewski for protocols.io.

    Written as used in Snead, Matyszewski, Dickey et al. 2022.

    Steps

    1.

    Typical reaction volume is 40µL .

    Add dye in 1:1 ratio to about 20micromolar (µM) .

    Note
    This protocol was used for LRRK1RCKW and LRRK2RCKW but should apply to other similar proteins and constructs as well.

    2.

    Incubate at Room temperature for 1h 0m 0s

    3.

    Remove excess dye by a buffer exchange through a Micro Bio-Spin P-6 desalting column (Bio-Rad).

    Make sure to equilibrate the column with the LRRK2 Storage buffer beforehand. Follow directions included with the column.

    3.1.

    Repeat the exchange one more time to further remove the excess dye.

    4.

    Quantify protein concentration and label efficiency using a NanoDrop or equivalent method.

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