pIMAGO Frequently Asked Questions

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How is pIMAGO different from phosphoprotein detection stains and other reagents?

Other “universal” stains for phosphoprotein detection are based on small molecules, with typically 1-2 binding groups and 1-2 detection groups per reagent. Each pIMAGO molecule, on the other hand, is a multi-functionalized dendrimer with multiple binding and detection groups per reagent. This ensures very strong binding to phosphoproteins that can survive harsh washing conditions (to improve specificity) and can be coupled with total protein antibodies for simultaneous multiplexed detection (many other products cannot survive the antibody binding binding). The multiple detection groups per pIMAGO reagent also amplify the signal from every binding event, thus significantly improving the detection sensitivity.

Can pIMAGO be used for in vitro kinase assays instead of radioactive ATP labeling?

pIMAGO can effortlessly replace radioactive ATP labeling for easy and safe in vitro kinase assays, eliminating the need for hazardous and expensive P32 experiments. However, because pIMAGO detects any phosphorylation event, a control needs to be run as well (e.g. reaction without ATP or kinase) to ensure the phosphorylation detected is from the tested reaction and is not endogenous.

Can I use the provided control phosphoprotein for quantification of phosphate in my own sample?

It is always difficult to do absolute quantification using different proteins. Various proteins may have different transfer efficiency for Western Blot or binding efficiency to a microplate. However, for relative quantification purposes, the amount of control phosphoprotein that we suggest loading contains approximately 16 pmol of phosphate.

How can I differentiate between different phospho-sites (Ser, Thr, Tyr)?

Unfortunately, there is no way to distinguish between different amino acids. As a universal reagent, it binds to all phospho-sites. The researcher can try using anti-pTyr general antibody as well. If anti-pTyr does not have any signal but pIMAGO does, then either Ser or Thr is phosphorylated.

Can I strip off the pIMAGO HRP signal to do subsequent antibody detection?

Yes. The pIMAGO signal can be stripped off using general harsh membrane stripping conditions. However, pIMAGO should always be bound first, before antibody binding.

How can I multiplex pIMAGO with antibody for simultaneous signal detection?

Because of the acidic nature of the pIMAGO buffer, antibodies cannot be incubated simultaneously with pIMAGO. However, there is a good way to detect both phospho and total protein signal simultaneously through sequential incubation procedure (i.e. pIMAGO binding and washing are carried out first before antibody binding). Suggested multiplexing procedure is as follows:

  1. a) incubate with pIMAGO
  2. b) incubate with total protein antibody
  3. c) incubate with avidin-Fluor680 and secondary antibody conjugated with Fluor-800 together (blocking buffer can be used for this step).

Both signals can be detected simultaneously using two different fluorescence detection channels.

What do you mean by universal detection of phosphoproteins?

pIMAGO is a nanopolymer-based reagent that binds to negatively charged phosphate groups on proteins. The carefully designed pIMAGO and washing buffers ensure that the binding occurs only with the phosphate groups and not other negatively charged molecules. Unlike antibodies, the binding and detection do not depend on the amino acid micro-environment. Therefore, any phosphorylation site on any protein can be detected, representing universal detection capabilities. The term also points to the ability of pIMAGO to detect phosphoproteins in many formats and surfaces (e.g. Western Blot, dot-blot, microplate, microarray, etc.).

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