Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel

Abstract

The sensitivity of Western blotting analysis after Phos-tag SDS-PAGE is occasionally inferior to that after normal (Phos-tag-free) SDS-PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos-tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet-tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos-tag SDS-PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos-tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet-tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos-tag gels were similar to those from normal gels. In some cases involving the use of a Phos-tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.

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“…Furthermore, this band shift was not seen when kinase‐dead Cdc5 was induced (Fig C). To verify the notion that these species corresponded to phosphorylated Dbf4, we resolved them on gels containing the Phos‐tag reagent, which specifically retards the migration of phosphorylated proteins (Kinoshita‐Kikuta et al , ). Consistently, the migration of Dbf4 was significantly retarded only when Cdc5 was induced, with increasing concentrations of Phos‐tag exaggerating this effect (Fig E).…”

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“…Membranes were transferred to Tris-buffered saline after the last wash and imaged within 1 h using a Li-Cor Odyssey scanner. Boxes were manually placed around each band of interest, which returned near-infrared fluorescent values of raw intensity with intralane background subtracted using Odyssey analytic software (27,31).…”

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“…Cell lysates were mixed with 3 × protein sample loading buffer (3.9 ml of 0.5 M Tris-HCl [pH 6.8], 0.6 g of SDS, 3.0 ml of 100% glycerol, 1.5 ml of 2-mercaptoethanol [2-ME], 1.5 mg of 0.02% bromophenol blue) according to the manufacturer’s instructions (Wako) and boiled at 100 °C for 10 min. The samples were resolved by Mn 2+ -Phos-tag SDS-8% PAGE containing 25 μM Phos-tag and 0.1 mM MnCl 2 as previously described 66 at a constant current of 25 mA at 10 °C for 7 h. After electrophoresis, the polyacrylamide gel was washed three times (40 min each) with transfer buffer containing 35 mM ethylene diamine tetraacetic acid (EDTA), followed by two washes (30 min each) with transfer buffer without EDTA according to the manufacturer’s instructions (Wako) with some modifications. Subsequently, the proteins were electroblotted onto polyvinylidene difluoride membranes (Millipore) at a constant current (270 mA) for 4 h. Immunoblotting was performed with a mouse monoclonal anti-GFP antibody (1:2,000; Abmart) as described above.…”