pIMAGO Frequently Asked Questions
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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.
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.
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.
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.
Yes. The pIMAGO signal can be stripped off using general harsh membrane stripping conditions. However, pIMAGO should always be bound first, before antibody binding.
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:
- a) incubate with pIMAGO
- b) incubate with total protein antibody
- 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.
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.).
PolyMAC Frequently Asked Questions
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50 µl of PolyMAC/beads mixture is designed for efficient enrichment of standard samples (e.g. 50-100 µg or less of peptide sample). For larger peptide amounts the PolyMAC beads volume and loading buffer volume should be increased accordingly. The optimal ratio should be tested for each sample type and could change depending on the amount of phosphorylation in the sample. There is no need to use less than 50 µl of PolyMAC/beads mixture for small sample amounts.
C18 desalting should be used for standard samples (e.g. typical cell lines or other cultures).
FASP should be used for for samples with lots of contaminants (e.g. plants or bacteria).
A combination of FASP + C18 desalting should be used for particularly “dirty” samples (large amount of lipids).
No extra clean-up is needed for clean samples in volatile solvents from IP or fractionation experiments.
100-ug starting material is suggested for highly phosphorylated samples (e.g. stimulated standard cell lines).
100ug-1mg starting material is suggested for yeast or unstimulated cell lines.
1mg starting material (FASP step recommended) is suggested for plants.
1mg-2mg suggested starting material is suggested for tissue samples.
100-200ug is a good starting point if the amount of phosphorylation is unknown.