Total protein staining

CBB G-250 is another popular total protein stain. Researchers blotting 2-D PAGE gels particularly favor it because it is compatible with mass spectrometry. Stained blots provide good media for archiving 2-D PAGE separations. A version of SYPRO Ruby, formulated for blots, is a very sensitive total protein stain. 

Check transfer efficiency by staining the gel after transfer, or by staining a second blot with a total protein stain, such as coomassie blue or ponceau red. Alternatively, use commercially available prestained protein standards that are run along the samples of interest and that are visible during both the separation electrophoresis and on the membrane after transfer... 

In all these methods proteins are transferred onto one membrane and both total protein staining and immunostaining are performed on the same membrane. Double-replica blotting methods have also been developed to obtain a membrane with all the proteins stained and that is an almost identical copy of the immunostained one. The first of its kind was described by Johansson (66) who found that by chang-... 

Deep Purple Total Protein Stain (Cat. No. RPN 6306, GE Healthcare). 

This green fluorescent Pro-Q Emerald 300 glycoprotein-staining method can he combined with SYPRO Ruby for total protein staining, with sensitivity up to... 

Sypro Ruby Total Protein Staining of Microarray... 

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Fig. 3.7 Transgenic rapeseeds were sprouted in an airlift tank with (lane 1) and without (lane 2) of streptomycin at 100 mg L-1. Total proteins were extracted, separated by SDS-PAGE and stained with Coomassie blue. The synthesis of Rubisco large and small subunits was inhibited as clearly shown in lane 2.

Fig. 8.4 Coomassie-stained SDS-polyacrylamide gel showing chloroplast transgenic lines expressing IFNa2b. Lanes 1 and 2 Total soluble protein (TSP) Lanes PH, 3 and 4 Total protein (TP).

For proper identification of the proteins of interest in a blot, immunodetected proteins must be compared to the total protein pattern of the gel. This requires the indiscriminate staining of all the proteins in the blot. Colloidal gold stain is... 

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

Figure 8.4 Effect of replacing small fraction of p-lactoglobulin by sodium caseinate on microstructure of concentrated oil-in-w7ater emulsion (45 vol% oil, 3 wt% total protein, pH = 6.8, ionic strength = 0.03 M) heated for 6 min at 90 °C. Confocal micrographs were obtained with Rhodamine B as fluorescent protein stain (a) emulsion contains 3 wt% p-lactoglobulin (b) emulsion contains 2.85 wt% p-lactoglobulin + 0.15 wt% caseinate. Scale bar = 20 pm. Reproduced from Parkinson and Dickinson (2004) with permission.

Proteins, blotted from polyacrylamide gels onto nitrocellulose sheets (Western blots) can be stained nonspecifically with a variety of dyes, or they can be identified individually by probing with appropriate antibodies. These procedures may be performed on duplicate blots, staining the total protein pattern on one blot and using the second blot for the immune reaction (1,2) This chapter describes how to combine both methods on one blot, i.e., staining the blot first for total protein, followed by an indirect immune reaction (3). 

Fig. I. Examples of Western blots stained with colloidal gold for total protein followed by immunostaining of individual antigensJ.anes 1—3 Proteins on a Western blot from a cytoplasmic extract of poliovirus-infected HEp-2 cells were stained with colloidal gold. The probing monoclonal antibodies, recognizing the viral proteins VP1 and precursor, VPO and VP2, and VP3, respectively, are detected by peroxidase-coupled rabbit-antimouse antibody (asterisks). Lane 4 Western blot of an E. coli lysate, containing a fusion protein composed of protein A and the poliovirus protein 2B. The fusion protein (arrowhead) is detected on the gold-stained blot by peroxidase-coupled IgG that binds to the protein A moiety.

Figure B3.1.2 Native discontinuous polyacrylamide gels activity stained for proteinases. (A) Gel stained with Coomassie brilliant blue for total protein. (B) Gel assayed for proteinase activity using casein as a substrate. Samples are enzyme extracts of hepatopancreas from four shrimp species. Lane 1, molecular weight markers Lane 2, Rcaliforniensis Lane 3 R vannamei Lane 4, Rpaulensis, Lane 5, P. schmitti.

Fig. 6. Under severe starvation conditions the epsi-APase and several other excreted proteins were present in the medium at very high levels relative to unstressed controls. Proteins excreted by cells growing 8 days -Pi or +Pi were separated via SDS-PAGE and immunoblotted using AP3 (A) or silver stained to show total protein (B). The psi enhancement of the epsi-APase (53.6 kDa) was clearly shown. The apparent molecular masses of several proteins selected for further study are indicated. Significant psi enhancement was shown for several of these proteins (Goldstein et al., 1989b).

Fig. 7. Evidence for protein phosphorylation as an early event in the psi response. 2 ml of three-day-old cells growing —Pi or +Pi were washed for 2 h in +Pi consisting of 20 pCi of carrier-free (32P)-orthophosphate. Cells were pelleted and total proteins extracted and separated via SDS-PAGE. A, Autoradiogram with equal cpm loaded per lane. The arrow indicates the position of a putative phosphorylated protein that showed psi enhancement. B, The same as A except that the lanes were loaded to represent equal amounts of cell biomass (dry weight). Dramatic enhancement in phosphorylation of all bands in B may be the result of increased rates of Pi transport in the —Pi treatment or these data may imply that several proteins show enhanced phosphorylation under psi conditions. C, As A and B except total proteins were visualised via silver staining. (All gels are aligned by apparent molecular mass.)...

Fig. 11.12. The use of gel microdroplets and flow cytometry to assay drug sensitivity of bacterial cells. The figure shows side scatter and green fluorescence contour plots of gel microdroplets (GMDs) containing E. coli cells that have been stained with fluorescein isothiocyanate for total protein. The microdroplets have been analyzed in the flow cytometer either at time 0 or 2 h after incubation in control medium (left plots) or medium containing penicillin (right plots). A model system was created by mixing two strains of bacteria (susceptible or resistant to penicillin). The data show that a small subpopulation of resistant cells could be detected within 2 h because of its rapid growth in comparison to susceptible cells. From Weaver et al. (1991).

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