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  • After electrotransfer of our proteins to a membrane, we will now block the

  • blot,

  • apply a primary antibody specific for our protein of interest and then a secondary antibody

  • which will recognize the primary antibody.

  • Start by removing the membrane from the cassette and rinsing three times in

  • water.

  • As an optional step, we can verify the proteins were transferred successfully

  • by staining the membrane with ponceau red.

  • Incubate the membrane in ponceau for five minutes and wash with water until

  • the bands are clear.

  • After verification the blot can then be de-stained by continuing to wash with

  • water or TBS twine until the dye is completely removed.

  • We need to block all areas of the blot which do not already contain protein.

  • This will prevent non-specific binding of the antibody and reduce overall

  • background signal.

  • Common blocking buffers include 5% non-fat dry milk for the assay

  • in a TBS-Tween solution.

  • However do not use a milk solution when probing with phosphor-specific antibodies as it can

  • cause high background from its endogenous phosphoprotein, casein.

  • Incubate the membrane with blocking solution for one hour at room temperature

  • under slight agitation.

  • Decant the blocking solution and wash with TBS twine for five minutes.

  • We are now ready to add our antibody. Dilute the primary antibody in a

  • blocking buffer at the concentration recommended on the datasheet.

  • Incubate overnight at 4 degrees Celsius with gentle shaking.

  • A recommended optional step is to also use a positive control loaded antibody

  • which allows the user to verify equal amounts of total protein were loaded into each

  • well and aides in troubleshooting by removing any uncertainties

  • with the Western Blot procedure. The next day, decant off the primary antibody and

  • wash the membrane with large volumes of TBS twine and vigorous agitation

  • five times for five minutes each.

  • These stringent washes are extremely important for removing non-specific

  • background signals.

  • After washing, dilute the secondary antibody in blocking solution and

  • incubate the membrane for one hour at room temperature at the concentration

  • recommended on the datasheet.

  • In our example the secondary is also conjugated to HRP for later

  • detection.

  • Decant membrane and wash secondary with large volumes of TBS twine with

  • vigorous agitation five times for five minutes each. You are now ready for the

  • detection phase.

After electrotransfer of our proteins to a membrane, we will now block the

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B2 中高級

Western Blot可視化協議。第4階段:免疫印跡 (Western Blot Visual Protocol: Phase 4: Immunoblotting)

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    Yu Sheng Chen 發佈於 2021 年 01 月 14 日
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