Hi, Ana. I am Eiji Kinoshita, who is one of developers of Phos-tag SDS-PAGE, at Hiroshima University in Japan. Thank you for your questions and using our Phos-tag reagent. Please find my answers described below. I hope they will be helpful for you.

1. How to efficiently preserve phospho-groups during IP reaction:

Why do you need the IP samples in the analysis using Phos-tag SDS-PAGE.

2.How to obtain a better resolution of bands using Mn-phos-tag system, since my protein is 122kDa:

The optimal concentration of Mn(II)–Phos-tag (e.g., 10-25 µM) and polyacrylamide (e.g., 4-6%) should be determined. In the case of less than 5% gel, I recommend using a polyacrylamide-agarose composite gel. Please see the following paper of Nature Protocols 4, 1513-1521, 2009 (PMID: 19798084).

Precipitation of cellular proteins:

We usually conduct Acetone precipitation, which is one of the general methods for precipitation of cellular proteins. The procedures are follows as:

1. Let acetone cool on ice before use.

2. Add four-fold amounts of acetone to the lysate sample.

3. Incubate the mixed sample for 2 h at –20 ˚C.

4. Centrifuge at 12,000 × g for 5 min.

5. Remove the supernatant fluid, and then dry the pellet*.

6. Re-dissolve the pellet with the next lysis buffer.

*Notice that over-drying pellet is very difficult to re-dissolve.

In our experiments, the pellet (precipitated cellular proteins) is usually re-dissolved with 1X Sample Buffer consisting of 65 mM Tris–HCl (pH 6.8), 2% LDS (lithium lauryl sulfate), 100 mM DTT, 10% glycerol, 0.01% BPB, and 6 M urea. Urea should be added just before use.

Because it is generally difficult to re-dissolve the precipitated proteins, the buffer with urea is used instead of normal Laemmli's Sample Buffer. Tthe sample boiling should be avoided when use the buffer with urea.

In the procedures described above, we have not felt the instability of phosphate group on Ser, Thr, or Tyr residue.

Good luck for your works!

Hi, I found this question because I want to precipitate the proteins of a cell lysate sample with the TCA/acetone method but I wanted to be sure to preserve the phosphorylated sites of the proteins. Here I see that precipitation with acetone leaves the aminoacid residues phosphorylated, dose TCA/acetone precipitation preserves the phosphate group on the residues as well?

Thank you!

I think the phosphate group on Ser, Thr or Tyr residue will be preserved, but that on the His or Asp residue, which is observed in two-component system for example, will not. That is dependent on your target. Thanks!

Were you successful, Ana? What percentage acrylamide did you find worked, if so?

Hi Joshua,

Unfortunately, I couldn't go with whole cell extract because I am working with the protein which is expressed in low level. Also, it turned out that my protein is highly unstable once purified.I did manage to isolate it only when I did the pulldown with its binding partner, but then I went to do a mass spec and resolved a phosho-map(I can give you more detailed protocol if you are interested). I can tell you that the protocol that I have used to prepare samples for mass spec  (after IP) is very similar to the one written by Eiji Kinoshita, and that the S/T sites are very well preserved, but you should avoid freeze-thaw cycles(I always used fresh samples). If you are working with less picky protein than mine, I would use 6% gel and try different concentrations of phos-tag. Bare in mind that if you are working with tagged protein, the linker peptide is frequent target of proteases, which may resolve as a lower band on a blot.

Good luck!