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    Column Buffer

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    Rana Mazhar Abbas
    The document provides a pilot protocol for purifying a protein using an amylose resin column. Key steps include growing E. coli cells expressing the protein, lysing the cells through freeze-thaw or sonication, centrifuging to separate soluble and insoluble fractions, mixing the soluble fraction with amylose resin to bind the protein, washing the resin, and analyzing samples on an SDS-PAGE gel to check purification. The column buffer contains Tris-HCl, NaCl, EDTA, TCEP, and glycerol to maintain protein stability during purification.

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    Column Buffer

    Uploaded by

    Rana Mazhar Abbas
    15 views2 pages
    The document provides a pilot protocol for purifying a protein using an amylose resin column. Key steps include growing E. coli cells expressing the protein, lysing the cells through freeze-thaw or sonication, centrifuging to separate soluble and insoluble fractions, mixing the soluble fraction with amylose resin to bind the protein, washing the resin, and analyzing samples on an SDS-PAGE gel to check purification. The column buffer contains Tris-HCl, NaCl, EDTA, TCEP, and glycerol to maintain protein stability during purification.

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    buffer

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    The document provides a pilot protocol for purifying a protein using an amylose resin column. Key steps include growing E. coli cells expressing the protein, lysing the cells through freeze-thaw or sonication, centrifuging to separate soluble and insoluble fractions, mixing the soluble fraction with amylose resin to bind the protein, washing the resin, and analyzing samples on an SDS-PAGE gel to check purification. The column buffer contains Tris-HCl, NaCl, EDTA, TCEP, and glycerol to maintain protein stability during purification.

    Copyright:

    © All Rights Reserved
    Download as ODT, PDF, TXT or read online from Scribd
    Download as odt, pdf, or txt
    15 views2 pages

    Column Buffer

    Uploaded by

    Rana Mazhar Abbas
    The document provides a pilot protocol for purifying a protein using an amylose resin column. Key steps include growing E. coli cells expressing the protein, lysing the cells through freeze-thaw or sonication, centrifuging to separate soluble and insoluble fractions, mixing the soluble fraction with amylose resin to bind the protein, washing the resin, and analyzing samples on an SDS-PAGE gel to check purification. The column buffer contains Tris-HCl, NaCl, EDTA, TCEP, and glycerol to maintain protein stability during purification.

    Copyright:

    © All Rights Reserved
    Download as ODT, PDF, TXT or read online from Scribd
    Download as odt, pdf, or txt
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    Column buffer

    20 mM Tris-HCl (HEPES is also ok)

    200 mM NaCl

    1 mM EDTA

    0.5mM TCEP is likely ok

    10 % glycerol is ok but not required

    Zinc sulfate is likely also ok


    Procedure
    Pilot protocol
    1. Grow a 5mL culture overnight in LB + 0.2% glucose + antibiotic

    2. Dilute back 100-fold into 80mL culture of LB + glucose + antibiotic

    3. Grow at 37C to an OD600nm of 0.6.

    4. Divide the cell cultures into two aliquots of 40 mL each.

    5. Harvest the cells by centrifugation at 4000 x g for 10 minutes.

    6. Discard supernatant.

    7. Resuspend pellet in 5mL of column buffer.

    8. Freeze the cells in a dry ice-ethanol bath.

    9. Thaw in icy water.

    10. Repeat freeze thaw cycles 2-3 more times to lyse cells.

    NEB recommends sonication for < 15 seconds while cells are in a ice water
    bath. Usually takes 2 minutes.

    Optional: monitor the release of the protein with a Bradford assay by adding
    10 L sonicate to 1.5mL of Bradford reagent and mixing.

    Save 13L cell lysate for SDS-PAGE gel.

    11. Centrifuge at 9000 x g at 4C for 20 minutes.

    12. Decant the supernatant and save on ice.

    Save 13L crude extract for SDS-PAGE gel.


    13. Resuspend pellet in 5 mL column buffer (insoluble fraction).

    Save 13L insoluble matter fraction for SDS-PAGE gel.

    14. Place 200 L of amylose resin in microfuge tube.

    15. Spin briefly in microcentrifuge.

    16. Resuspend the resin in 1.5mL column buffer.

    17. Microcentrifuge briefly.

    18. Discard supernatant.

    19. Resuspend the resin in 200 L column buffer.

    20. Mix 50 L crude extract with 50 L amylose resin slurry.

    21. Incubate 15 mins on ice.

    22. Microcentrifuge for 1 minute.

    23. Remove supernatant and discard.

    24. Wash pellet with 1mL column buffer.

    25. Microcentrifuge for 1 minute.

    26. Remove supernatant and discard.

    Save 13L of amylose bound to protein for SDS-PAGE gel.

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