Hi,
I tried to analyze the phosphorylation status of my protein with the PhosTag acrylamide method. For this I lysed my HEK cells with RIPA buffer (50 mM HEPES-KOH, 150 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.1% SDS, 0.5% Sodium deoxycholate, cOmplete Prot. Inhibitor, PhosSTOP). Because it is recommended in the PhosTag user guide I eliminated EDTA with TCA precipitation.
I run 10% acrylamide, 50 µM PhosTag gel but can't detect any shifts.
I realized, that TCA might be not a good idea for His-phosphorylated proteins, does anyone has an idea, how I can prepare my samples to suit the PhosTag gel recommodations?
You could also try precipitating with acetone instead of TCA. However, you will have to use some precipitation method otherwise your sample will not run properly on the PhosTag gel. Do you have controls like non-phosphorylated or de-phosphorylated with phosphatase?
Thank you for your answer!
Is it possible to lyse the cells without EDTA? Anyway, I will try acetone precipitation.
I thought about dephosphorylating my sample with a commercial phosphatase, so far I didn't, can you recommend a phosphatase?
You can lyse cells without EDTA. EDTA mostly protects your sample from degradation. EDTA also helps break the nucleus. However, EDTA is not the only thing that messes up how samples run on PhosTag gels. The other salts (like NaCl) will also mess up how the samples run. Precipitation is necessary in most cases to get nice bands on PhosTag gels.
You would probably need a histidine phosphatase (I do not know much about this subject). Perhaps you could use acid to dephosphorylate your samples as well (maybe acetone precipitation vs TCA precipitation). This older paper may help you figure out how to dephosphorylate your samples: http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1033.2002.02755.x/pdf
Hi, you should avoid any chelating agent such as EDTA that can chelate either Zn2+ or Mn2+. Also, include benzonase in the buffer to break down DNA/RNA which can cause smears on the Phos-tag gel. Finally, it might be helpful to have a known phosphorylated protein as a control.
Good luck
I recommend trying a simpler approach. First of all, you do not want EDTA to chelate the Zn or Mn ions from the Phos-tag gel, which is why it is typically removed/excluded from sample preps. If TCA is not compatible with His-phosphorylation, then I would just skip this step and run the samples directly on the gel. You can prevent the chelation of the ions from the gel by adding 1-2 mM MnCl2 (assuming you are using the Mn2+ Phos-tag gel chemistry) in your sample prep when you dilute with your Laemmli buffer [this was recommended in the Phos-tag gel guidbook].
Additionally, you can also prevent ion chelation from the gel by increasing the amount of MnCl2 you add to the gel when you cast it. I use 300 uM and this has given me the best results. So try making a 10% gel with 50 µM PhosTag and 300 uM MnCl2.
Lastly, heating the samples to high temps (>95C) has significantly reduced the phosphorylation of my proteins. I recommend never heating your samples too high. Instead, during the denaturing/reducing heating step with Laemmli buffer, heat to 65C for 10 min.
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