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TCA protein precipitation protocol

By Anne Lawrence,2014-02-01 23:54
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TCA protein precipitation protocol

1. TCA protein precipitation protocol

    (orginally from Luis Sanchez)

    revised: October 10, 2001

    author: clw

    Stock Solutions: 100% (w/v) Trichloroacetic acid (TCA)

    recipe: dissolve 500g TCA (as shipped) into 350 ml dH2O, store at RT. (for details, check Maniatias under TCA ppt )

    Precipitation Protocol:

    1. Add 1 volume of TCA stock to 4 volumes of protein sample. i.e. in 1.5ml tube with maximum vol., add 250μl TCA to 1.0ml sample.

    2. Incubate 10 min at 4?C.

    3. Spin tube in microcentrifuge at 14K rpm, 5 min.

    4. Remove supernatant, leaving protein pellet intact. Pellet should be formed from whitish,

    fluffy ppt.

    5. Wash pellet with 200μl cold acetone.

    6. Spin tune in microfuge at 14K rpm, 5min.

    7. Repeat steps 4-6 for a total of 2 acetone washes.

    8. Dry pellet by placing tube in 95?C heat block for 5-10 min to drive off acetone. 9. For SDS-PAGE, add 2X or 4X sample buffer (with or without bME) and boil smaple for

    10 min in 95?C herat block before loading smaple onto polyacrylamide gel.

2.

    Add an equal volume of 20% TCA (trichloroacetic acid) to protein sample. Incubate 30 min on ice.

    Spin in microfuge at 4 deg. For 15 min.

    Carefully remove all supernatant.

    Add ~300 ul cold acetone and spin 5 min at 4 degrees.

    Remove supernatant and dry pellet.

    Resuspend samples in SDS PAGE loading buffer. Load to SDS PAGE after heating at 65 deg for 3 min.

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