Recently I've use Phos-tag PAGE to detect the phosphorylation form of my protein of interest. However, there is only one band and that makes me wonder if the protein extraction method i use causes the degradation of phosphorylated protein... Here is my protocol (i've checked there's no EDTA in any of buffers)
1. Grind plant tissue with liquid N2
2. cast protein extraction buffer (100mM Tris-HCL, 3M urea, 5% SDS, 10% glycerol)
3. heat extraction buffer at 100C for 3 min and add protease inhibitors (2ng/ml apotinin, 3ng/ml leupetin, 1ng/ml pepstain, 2mM PMSF and EDTA-free protease inhibitor cocktail)
4. add boil extraction buffer to lysate, mix by vortex
5. heat samples at 100C for 3min, followed by centrifugation
6. measure the protein concentration of supernatant
7. add 6x sample buffer (375mM Tris-HCl, 10% SDS, 50% glycerol, 10% 2ME, 0.03% bromo blue) to samples and perform phos-tag PAGE
Attached is my result, my target protein is 55kD and I used 12% acrylamide and 50umol/l phos-tag. There are several things I might adjust in my next-try, based on many wonderful suggestions from previous discussions about Phos-tag PAGE.
1. use 8% instead of 12% acrylamide gel
2. longer running time
3. use 25 uM but not 50uM phos-tag
So..should I switch my protein extraction method to TCA extraction method? is there other idea to improve the experiment?
ANY suggestion is welcome!
Gloria, in my experience with proteins phosphorylated at histidine residues, boiling might not be a very good idea since it may favor the hydrolysis of the phosphate bond. I've used buffers with EDTA to quelate Mg, since it has been reported to be important for the dephosphorylation reaction. Particullarly for analyzing proteins from bacteria, we use physical lysis methods (such as Fastprep homogenizers) to avoid hydrolysis problems with chemical methods. However, I don't know if it might work with plant tissues. I hope it might have helped!
Hi Ignacio! Since I don't know the phosphorylation sites of my protein, I should try to preserve all of them. Thanks!
As mentioned by Ignacio, if your target is a His- or Asp-phosphorylated protein, you should avoid the heat treatment. In my analysis of the bacterial two-component system, all the up-shifted bands detected on the Phos-tag gel disappear through the heat treatment of samples (at 95 °C for 3 min). The phosphoryl groups on His and Asp residues are chemically unstable. So, TCA method is so bad for these phosphorylated proteins. In the case of analysis of Ser-, Thr- or Tyr-phosphorylated protein, however, the heat treatment usually does not have any problem. Which type of phosphorylation on your target protein?
I assume that your sample preparation has not any problem, and I describe below general suggestions.
I think there are two possibilities for only one band.
First, your target was not phosphorylated by the treatment you did under the experimental conditions, because there is no up-shifted band on the Phos-tag gel. Have you ever checked the phosphorylation of your target by using any other methods before?
Next, Phos-tag did not work under your experimental protocol, but I don’t really understand that reason.
So, I think you should use a phosphorylated positive control, which shows surely a shift in the mobility on your Phos-tag gel, and the corresponding dephosphorylated negative control.
I do hope my suggestions help your works. Good luck!
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