I am studying phosphorylation of a gene of my interest. In convenience, I call it “gene A.” At first, I have found that a threonine residue followed by proline in “gene A” is phosphorylated under specific condition via proteomic analysis using Flag-tagged “gene A” immunopurified from human cells. So, we have constructed its Flag-tagged mutants whose threonine residue is converted into alanine (hereafter, A form) or aspartate (hereafter, D form) to mimic its un-phosphorylated or phosphorylated status, respectively. Using these mutant series, I found its probable phosphorylation affect its interaction with other cellular partners. To extend such findings to physiology, then, I decide to confirm whether “gene A” could be really phosphorylated in human cells under the specific condition.
To do this, I first transfected Flag-tagged WT or A form of “gene A” for Flag-immunoprecipitation to survey its phosphorylation via Western blotting using phosphor-specific antibodies. Two kinds of phosphor-specific antibodies were applied: rabbit anti-phospho-threonine antibody (isotype IgG) purchased from Abcam (cat No. ab9337) and mouse anti-phospho-threonine-proline antibody (isotype IgM) obtained from Cell Signaling (cat No. #9391), which can detect phosphorylated threonine residue followed by proline in a context-independent manner. However, I was worried that the size of “gene A” is ~50 kDa. Furthermore, its Flag-tagged version is ~55 kDa. Immunoglobin, particularly heavy chain, in the bound fraction from Flag-immunoprecipitation could strongly hide what I want to know from Western blotting using those phospho-specific antibodies.
Unfortunately, REALLY did it happen. When I performed Western blotting, strong chemiluminescence signal from heavy chain always encroaches possible existing signal from phosphorylated “gene A”, regardless of secondary antibodies such as anti-rabbit IgG (H+L), anti-mouse IgG (H+L), anti-mouse IgM (H+L), etc. They all strongly detect heavy chains, and anti-rabbit IgG (H+L)-HRP also strongly detect mouse heavy chain. Now I think that, therefore, Western blotting using phospho-threonine-specific antibody is not applicable for detecting phosphorylation of “gene A”.
Next, I tried Wako Industry’s Phos-tag gel where phosphorylation status of a specific gene can be separated during conventional SDS-PAGE and easily detected via conventional Western blotting using specific antibody raised against that specific gene. This system makes phosphorylated protein move slower than unphosphorylated form, permitting someone to examine phosphorylation of a gene in a mobility-dependent manner. Undergoing several trials and errors, I could see that the “threonine residue” is really phosphorylated, when comparing between WT and A form. That is, A form moved faster than WT during SDS-PAGE. However, the phosphorylated form emitted much weaker signals than unphosphorylated form during Western blotting. In other words, strong signal from major unphosphorylated bands completely burned out X-ray film before signal from phosphorylated “gene A” make seemingly band pattern.
In summary, I have a confidence that the threonine residue in “gene A” is really phosphorylated, but I have no clear-cut and figure-quality data to argue that this residue is really phosphorylated in physiological condition, due to experimental limitation and its seemingly weak phosphorylation.
How can I overcome such problems? Is there any methodology to show the phosphorylation of a gene except for strategies mentioned above?
The phosphorylation at a Thr-Pro will cause a bit of a mobility shift alone. You could run the protein to the bottom of an 8% gel and see the shift. Check Bruno Fonseca's papers with his nice p70 S6 kinase band shifts for a nice protocol. You can also try 2D gel electrophoresis for some proteins it works perfectly. Also look at the database phosphosite.org someone probably already proved the site exists under physiological conditions by mass spec.
To avoid your detection problem you can try secondary antibodies which only recognize light chains from first antibody or native antibodies (Clean-Blot, EasyBlot).
Here are same examples of such antibodies:
http://www.piercenet.com/product/clean-blot-ip-detection-reagents-kit
http://www.genetex.com/Web/News/NewsList.aspx?id=269
Alternatively you can try to clone GFP-fused protein. This would increase the size of your protein and enable a very efficient purification via GFP-trap-beads.
In general, it is possible that only a small amount of your protein is phosphorylated under conditions you used for analysis. Variations can arise from different cell cycle phases (G1 vs. G2/M), mitogen or stress signals. Since your site is proline-associated, then most probably proline-directed kinases are responsible for this phosphorylation. There are four families of such kinases: CDK, MAPK, GSK3 and CLK. You can try to stimulate them to increase the phosphorylation of your protein.
you can give a try for phos and non-phos control in a western, usually phos moves differently from non-phos, it's the quick way to find it out. certainly, you can use specific antibodies
A gene is a sequence of nucleotides - how do you want to phosphorylate this??
1. You are not looking at a gene ... its a gene product ... a protein. You can run the primary sequence through a bioinformatic tool at the Expasy site (http://www.expasy.org) that identifies putative phosphorylation sites for various protein kinases. Its not fool-proof, but it can help.
2. Following on Eugen Werwein's suggestion. Pull down your protein using the epitope tag. Incubate it with activated, proline-directed protein kinases plus gamma-32P-ATP (10-100 uM) plus 10 mM MgCl2 and phosphatase inhibitors. Resolve on SDS-PAGE. Dry Gel. Expose to film (similar to ECL). If your facility does not permit the use of radioactivity, you can probe with anti-phospho antibodies, but 32P is the most sensitive approach. Several companies sell activated kinases - but the are quite expensive. About the same cost as an antibody. You may be able to collaborate with a lab that has expertise and reagents for working with protein kinases.
Thank you all for your helpful comments. "gene A" should be corrected into "protein A". It's my incorrect writing. I'll try further as your suggestions. Thank you.
I might have got that wrong, but why bother immunoprecipitate (hence the heavy chain IgG) at the time when you detect phosphorylation? Isn't a western blot (total protein vs phospho protein) enough?
In case not you can also avoid the heavy chains by using cross linked antibody-beads. There is a protocol for crosslinking them, you can also obtain crosslinked ones (in your case OctA beads from santa Cruz, used them in flag IP and didn't leave any hints of Ig)
http://www.scbt.com/datasheet-807-octa-probe-d-8-antibody.html
From my past experience, phostag technology could require extensive protein-specific standardization. As many people mentioned, you can avoid the antibody signals in the IP by several approaches (discussed in detail in some other researchgate topics before)
1. use of LC specific secondary / conformation specific secondary
2. you can cross link antibodies on beads and in case you are already using Flag-beads from sigma, u need to elute the protein in non-reducing conditions to avoid antibody elution
Another possible option (only if your protein is strongly overexpressed and phosphorylated) is to run transfected (wt and S-Amutant) and non-transfected samples and probe the lysates with pT and antiFlag antibody. The pT antibody will certainly give a lot of bands and mostly a smear, but If you can detect a unique band in the wt protein transfected lysate alone with the phospho-antibody (at the size of Flag-signals ), then it could be OK. You may still need further verification (may be u can immunodeplete this band with Flag beads to further prove the identity).
Good Luck