Plusone silver staining kit

COS-7 cells were transfected with GFP-CaMKIγ or GFP-CaMKIγ-C474S and Myc-CaMKKactive or an empty vector using Lipofectamine 2000. After transfection for 48 h, an immunoprecipitate kinase assay was performed using MBP as a substrate (Takemoto-Kimura et al., 2003 (link)). CaMKIγ immunopurification was evaluated by silver staining using a PlusOne Silver Staining Kit (Amersham). CaMKK expression was functionally confirmed by the similar intensity of the phosphorylated CaMKIγ band under extended exposure.

To visualize protein electrophoresis, silver staining was carried out using PlusOne Silver Staining Kit (Amersham biosciences). Stained-2DE gels were scanned using PowerLook 1100 (UMAX, Korea). Comparative image analysis was carried out using Progenesis software (Nonlinear Dynamics, Newcastle, UK). Gels were stained with silver to select spots of interest. Meanwhile, Coomassie brilliant blue staining was performed for spot identification by mass spectrometry. For protein identification, proteins in gel were fixed by soaking in a solution containing 40% (v/v) methanol and 10% (v/v) acetic acid for 1 h with gentle agitation. Fixed gels were stained by colloidal Coomassie staining solution (ProteomeTechInc, Korea).

After electrophoresis, the gels were stained with MALDI TOF compatible silver nitrate using a Plus One Silver Staining Kit (Amersham Biosciences), as described by Shevchenko et al. [26 (link)]. Gels were scanned with an ImageScanner II Desktop (Amersham Biosciences) and the image analysis of the gels was performed using PD Quest 8.0.1 software (Bio-Rad). Three gels were produced from independent cultures of each condition and representative image gels are shown. Induction factors for each induced protein were calculated as the ratio between the normalized value in “treated” gel and the normalized value in “control” gel. Protein spots displaying ≥2-fold change in abundance were chosen for analysis by mass spectrometry.