Early exploration of unmapped biological signaling pathways were carried out using radiolabeled phospho-imaging. The development of phospho-specific antibodies to detect and quantify protein phosphorylation made life easier for researchers (less 32P waste to deal with), but the interpretation of data from these experiments comes with its own set of caveats.
A balance of cascading kinases and opposing phosphatases determine when and where signals are propagated by adding and removing phospho groups, respectively. Protein phosphorylation modifications were first observed to occur at Ser/Thr and His/Asp residues, and tyrosine phosphorylation was discovered subsequently. These protein modifications relay information from protein to protein in vast, interconnected networks, and touch just about every important biological function from cell cycle to metabolism, from nervous system function to immune responses, and from tissue development to cancer.
To follow the signal, you want to know the phosphorylation state of your target protein. Antibodies that recognize peptide motifs in the phosphorylated state are valuable research tools in western blotting (WB) and in other applications. These range from antibodies that broadly recognize phosphorylation in many proteins (for example, phospho-tyrosine), to those that recognize PTM motifs defined (with varying degrees of degeneracy) by neighboring residues, to those that are specific for a PTM in a specific peptide. For most scientists pursuing a signaling hypothesis, peptide-specific phosphoprotein antibodies are the tool of choice when they are available.
When using phosphoprotein antibodies in WB, one must keep in mind the rarity of these modifications, appreciate the fragility of the phosphopeptide (phosphoester) bond, and be aware of the risk of artefactual dephosphorylation by prowling phosphatases. Cavalier laboratory practices make it harder to detect your phosphoprotein, whether by loss of the phospate group (phosphatases not inhibited), protein degradation (proteases not inhibited), or by reducing the effectiveness of antibody detection.
Here are some considerations to keep in mind:
• To ensure your target phosphoprotein is still phosphorylated and not degraded, include phosphatase and protease inhibitors (see CST #5872) to the lysis buffer. You may also want to consider kinase inhibitors (1).
• Work fast, keep it cold and sonicate to obtain total cell lysis (see Western Blot protocol). The speed of tissue procurement and treatment, cooling of the sample and thorough disaggregation are essential to minimize phosphatase/protease activity and to preserve your signal (1-5).
• Pay attention to pH. Although phosphoserine, phosphotyrosine, and phosphothreonine bonds are stable in non-physiological pH, antigen recognition may be negatively impacted by changes in pH for some antibodies. In addition, electrophoretic resolution and membrane transfer can be adversely affected if the pH is off in the running or transfer buffers, respectively.
• CST recommends using 5% Nonfat Dry Milk #9999 in 1X TBS, 0.1% Tween® 20 to block the membrane prior to introduction of antibodies. We do not see background nor cross-reactivity with milk proteins, including casein. With regards to phosphatase activity, the pasteurization process kills all alkaline phosphatase activity in milk.
• Check the product webpage/datasheet for recommended antibody dilution. Note that while the membrane is blocked with nonfat dry milk, phospho-specific antibodies are often diluted and incubated with the membrane in 5% BSA.
• Use TBS-T rather than PBS-T in wash/incubation buffers since the phosphate ion may lead to interference.**
• If you plan on stripping and re-probing the blot, probe for the phosphoprotein first since the stripping process often leads to loss/degradation of the phosphoprotein antigen. Due to the likelihood of low abundance of the antigen, CST recommends incubation overnight at 4°C with the primary anti-phosphoprotein antibody (see the CST Western Blot Troubleshooting Guide). The blot can then be stripped and re-probed with an antibody against the total protein, which serves as a loading/transfer control.
• Multiplexing fluorescent reporters (see the Fluorescent Western Protocol) can allow both the total protein and the phosphoprotein to be measured simultaneously, provided you have the imaging capability.
• CST recommends using a positive control to confirm that detected bands are phosphorylated. The use of specific activators and inhibitors to manipulate activation of signaling proteins, or comparison of blocking with phosphorylated vs. nonphosphorylated epitope peptides may also be employed. Phosphatase treatment is commonly used as a negative control to complement other experimental perturbations.
• Phosphoproteins are frequently in low stoichiometric abundance. A null result can and probably should be verified by concentrating the antigen by immunoprecipitation.
Finally, if all the experimental details are optimized, you should be able to detect that elusive phosphoprotein, at least one phosphorylation site at a time. However, multiple phosphorylation sites may well be present, leading to additional levels of complexity. Then, it might be time visit your local mass spectroscopy core facility, or call a CST scientist about using our PTMScan® service.
**You might be wondering about the use of PBS in other protocols, namely immunofluorescence and flow. In those protocols, macromolecules are crosslinked with aldehydes, and the primary amines present in tris (component of TBS) can interfere, making PBS advantageous. The value of PBS vs. TBS in WB has historically been somewhat contentious, and may be antibody/epitope specific.
- V. Espina et al., A portrait of tissue phosphoprotein stability in the clinical tissue procurement process. Mol Cell Proteomics 7, 1998-2018 (2008).
- K. A. David et al., Surgical procedures and postsurgical tissue processing significantly affect expression of genes and EGFR-pathway proteins in colorectal cancer tissue. Oncotarget 5, 11017-11028 (2014).
- A. S. Gajadhar et al., Phosphotyrosine signaling analysis in human tumors is confounded by systemic ischemia-driven artifacts and intra-specimen heterogeneity. Cancer Res 75, 1495-1503 (2015).
- S. Gundisch et al., Critical roles of specimen type and temperature before and during fixation in the detection of phosphoproteins in breast cancer tissues. Lab Invest 95, 561-571 (2015).
- A. P. Theiss, D. Chafin, D. R. Bauer, T. M. Grogan, G. S. Baird, Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation. PLoS One 9, e113608 (2014).