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I need to detect a FLAG tagged protein on a western blot and have to order an antibody. I'm not expecting to see a massive amount of my protein. To keep analysis time to a minimum I am considering a HRP-conjugated anti-FLAG. Are there any disadvantages to it (e.g. sensitivity)? First hand experiences/problems?

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  • $\begingroup$ Welcome to BioSE. It would be better if you make your question more precise? Disadvantage compared to what? Have you tried an experiment yet? $\endgroup$ – WYSIWYG Mar 27 '15 at 12:16
  • $\begingroup$ Compared to the classic incubation with primary anti FLAG or HA and subsequent detection with a secondary antibody coupled to HRP (e.g. anti goat/rat/mouse) $\endgroup$ – mimat Mar 27 '15 at 12:31
  • $\begingroup$ The obvious problem is signal amplification as pointed out in the answer. I don't have a first hand experience but I think that chemiluminescence based substrates would be better for a one-step WB. $\endgroup$ – WYSIWYG Mar 27 '15 at 13:31
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You can compare directly coupled antibodies to the classical indirect primary-secondary systems:

Directly coupled antibodies

Advantages:

  • quicker workflow as only one antibody is used
  • eliminates the chance of a cross-reacting secondary antibody (possibly less background)
  • possibility to use differently labelled antibodies on the same blot

Disadvantages:

  • lower sensitivity because the signal is not amplified
  • only few labels are available
  • directly labelled primary antibodies tend to be expensive and you need relatively high amounts of them
  • the labelling can damage or reduce the immunoreactivity of the antibody

Indirect Primary-Secondary Antibody systems

Advantages:

  • the secondary antibody amplifies the signal (which is especially interesting for weakly expressed proteins)
  • differently labelled secondary antibodies can be used on the same primary
  • primary antibodies from different species can be used simultaneously
  • one secondary antibody can be used with different primary antibodies (for example for the protein of interest and the loading control)
  • the labelling reaction does not affect the primary antibody

Disadvantages:

  • cross-reactivity of the secondary antibody can lead to non-specific bands
  • the method takes longer, as more steps in the process are necessary

For me the most important point is the higher sensitivity of the indirect method, for comparision see these images (from Life technologies):

enter image description hereenter image description here

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  • $\begingroup$ Thank you for confirming my suspicions! Primary-Secondary it will be then. $\endgroup$ – mimat Mar 27 '15 at 14:49

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