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ATM is a high molecular weight protein, so we use 3% polyacrylamide gel strengthend with 0.5% agarose gel.
HeLa cells were cultured in DMEM without serum, and treated actinomycinD (2µM) for 5 hrs.
Cells were lyzed by SDS-PAGE loading buffer.
In Zn2+–Phos-tag SDS-PAGE using the neutral-pH gel system buffered with Bis-Tris–HCl , on the other hand, we found that there was a limitation in the preparation of such highly porous gels, in that Bis-Tris-buffered gels did not have adequate sieving properties at concentration of less than 4% w/v polyacrylamide. Because Bis-Tris is a weakly basic amine (pKa6.5 at 20˚C) that is
partially protonated at neutral pH values, the free amine species (R3N) is converted into an amine radical cation(R3N+) that can act as a radical quencher during polymerization of acrylamide in the presence of ammonium persulfate. Therefore, the abundant Bis-Tris molecules interfere with the formation of a polyacrylamide gel matrix at very low concentrations of acrylamide. To overcome this limitation, we adapted a neutral-pH gel system buffered with Tris–AcOH for phosphorylation profiling of high-molecular-mass proteins by Zn2+–Phos-tag SDS-PAGE.
To demonstrate the utility of our newly adapted Tris–AcOH system, we performed a separation analysis of ATM with a molecular mass of 350 kDa by Zn2+–Phos-tag SDSPAGE.
HeLa cells were treated with 0 mM (control) or 2 mM actinomycinD for 2hrs, and lyzed with SDS-loading dye.
Tris–AcOH buffer Zn2+–Phos-tag gel produced a single band in the case of the control sample (left lane: –) and three additional up-shifted bands in the case of the ActinomycinD treated sample.
On the other hand, Mn2+–Phos-tag gel strengthened with agarose produced only two up-shifted bands corresponding to phosphorylated ATM .
Therefore, the currentZn2+–Phos-tag SDS-PAGE using the Tris–AcOH buffer (pH 7.0) permits a more-detailed detection of shifts in the mobility of phosphorylated ATM in response to DNA
damage than does the previous Mn2+–Phos-tag SDS-PAGE.
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