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General

The forced proximity test helps to assess the quality of the biotinylated antibody. If it does not pass, then either the antibody is under-biotinylated, or there still is free biotin in the solution. Even as little as 80 nM free biotin can cause the test to fail. Make sure to dialyze the antibody well (change the buffer at least five times, including one overnight exchange). We recommend using the Slide-A-Lyzer® Mini Dialysis Unit. Repeat the dialysis steps and perform the test again.

If your protein target is not on the list, you can create your own antibody probes with the antibody of your choice using the TaqMan® Protein Assays Open Kit. If your antibody is not biotinylated, you will have to biotinylate it before use with the Open Kit.

There may be several reasons for not seeing any change in expression. Check for the following:

  • Did the antibody probes pass the forced proximity test?
  • What is being used for the negative control? If there is endogenous expression of the target, consider using buffer instead of a “negative” lysate.
  • Make sure to test a range of inputs, using 2- to 3-fold dilutions
    • For cells: 1–500 cells
    • For recombinant protein:  ~200 pg/well
    • For total protein (tissue): 1–1,000 ng
    • For cell lysate: ~50 ng total protein/well
  • Make sure to use a positive control to ensure detection
  • Test with one of the control assays, such as for hCSTB or hICAM1

There may be several reasons for not seeing any change in expression. Check for the following:

  • Make sure to test a range of inputs, using 2- to 3-fold dilutions
    • For cells: 1–500 cells
    • For recombinant protein: ~200 pg/well
    • For total protein (tissue): 1–1,000 ng
    • For cell lysate: ~50 ng total protein/well
  • -Make sure to use a positive control to ensure detection
  • Test with one of the control assays, such as for hCSTB or hICAM1
  • Try optimizing some of the conditions, such as:
    • Modifying the binding reaction time and temperature (25°C for several hours, or 4°C overnight)
    • Reducing the assay probe concentration in the binding reaction (from 500 to 50 pM)
    • Decreasing the prox-oligo volume
  • Try swapping the two antibodies with the 5’ and 3’ prox-oligos. Sometimes the different orientation of the antibodies and oligos can help.

We recommend using one of our control assay probes to make sure that the system is working before testing with your own antibody probes. We have two options to choose from, which are expressed in most common cells lines: hICAM1 (Cat. No. 4405471) or hCSTB (Cat. No. 4405465).

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