Colloidal coomassie staining

If external calibration of the spectrum is to be used, a spectrum of the calibration standards should be acquired immediately, using the same power setting. Figure 1 shows a typical MALDI spectrum obtained from the trypsin digestion of a weak colloidal Coomassie-stained ID gel band. 

Make up protein samples [80-100 pg of radioactively labeled proteins, 3 x 50 pg of DIGE-labeled proteins (see Note 5), or 350-600 pg of unlabeled proteins for Colloidal Coomassie staining) (see Note 6)] to 360 pL with 8 M urea/2 M thiourea. Subsequently, add 40 pL of lOx rehydration buffer and mix the solution by shaking at room temperature for 30 min. Centrifuge the rehydration mix for 5 min at 21,000 x g (20 °C) to remove insoluble proteins. 

SDS-PAGE gels (Excel 8-18% gradient and Excel 15% homogeneous gels) were purchased from Pharmacia and electrophoresis was performed on a horizontal Pharmacia Multiphore II electrophoresis unit. The gels were either silver or colloidal Coomassie stained. 

Protein content as estimated from colloidal Coomassie stained SDS-gel. 

Stain the gel with Colloidal Coomassie staining solution. For this, immerse the gel in Coomassie staining solution and incubate by gende rocking at room temperature until bands become visible (usually 3-6 h or overnight). Rinse the gel 2-3 times with water and store if desired in 20-25 % (w/v) (NH4)2S04 in water (wNote 10). 

Ideally, the gel should have been stained with a Coomassie stain—preferably a colloidal Coomassie if detection sensitivity is an issue, e.g., (2,3), or one of the commercially available stains. This method is also compatible with Sypro stains (though an ultraviolet transilluminator will be required to visualize the protein bands or spots during excision). Standard silver stains are not compatible, but certain modified silver stains—e.g., (4), or one of the commercially available mass spectrometry compatible silver stains—can be used however, even these usually give inferior mass spectrometry data compared to Coomassie- or Sypro-stained gels. 

Figure 1 Reference 2-D PAGE map of rat serum. Unfractionated rat serum (30 jxl) was analyzed by 2-D PAGE. The separation was based on a pH 4-7 linear IPG in the first dimension and 11-18% SDS-PAGE in the second dimension. Proteins were visualized by staining with colloidal Coomassie. Some of the major proteins of rat serum are labeled.

Application of constant voltage 150 V to the gel for 50 min at room temperature Gel stained by colloidal Coomassie Blue, 1-cm3 pieces protein bands excised from the gel slab... 

The formation of TEPs may transfer more than just reactive carbon to the ocean ecosystem. As Mari [85] has pointed out, the composition of TEPs can be influenced by the adsorption of amino acids [92] and metals [93]. In addition, Long and Azam [94] have identified Coomassie stained particles (CSPs) as protein-rich aggregates in seawater. This means that, even though the process of colloid aggregation can exert a strong influence on the cycling of bio-... 

For identification, protein spots were cut from colloidal Coomassie Brilliant Blue-stained gels equivalent to the gel shown in Fig. 14B. Excised... 

Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., Orecchia, P., Zardi, L., and Rigetti, P. G. (2004) Blue silver a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25, 1327-1333. 

Note The elution should be optimized by checking at least two to three different elution solvents. Eluates may be assessed using a one-dimensional (ID) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel stained with colloidal Coomassie. 

Fig, 3.14. (a) 2D BN-PAGE of bovine serum stained with colloidal Coomassie. (c) S, Cu and Zn bonded to the protein were detected by (b) LA-ICP-MS In five line scans from the (BSA) protein band. 

Alternative staining procedures (reviewed in refs. 1—3) utilize Coomassie blue, Ponceau S, Amido black, India drawing ink, colloidal gold, or silver. A highly sensitive technique utilizing eosin Y has also been described (18)... 

Neuhoff, V., Stamm, R., Pardowitz, I., Arold, N., Ehrhardt, W., Taube, D. (1990). Essential problems in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels, and their solution. Electrophoresis 11,101-117. 

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