Coomassie blue staining

For visualisation of proteins after separation on gel, one could use different stains such as Coomassie blue stain or more sensitive silver staining. The Coomassie blue staining is relatively less sensitive than silver staining, but is highly convenient to use. 

The dye Coomassie Brilliant Blue R250 nonspecifically binds to all the protein. The gel is soaked in the dye for it to seep in and bind to the proteins. The gel is then destained to remove the unbound dye. The dye binds to the protein and not the gel, and hence the protein bands can be visually seen. The binding of the dye to the protein is approximately in stoichiometry, so the relative amounts of protein can be determined by densitometry. For most SDS and native gels, separated proteins can be simultaneously fixed and stained in the same solution. 

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A) Proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining. Lane 1, 4 )ig purified PGl Lane 2, 2 pg purified PG2 Lane 3,2 pg purified subunit. 

Fig. 13.2. The peptide components of H-gal-GP and TSBP visualized by Coomassie Blue staining of non-reducing (lanes 1 and 3) and reducing (lanes 2 and 4) SDS-PAGE gels.

Densitometric scans of Coomassie Blue stained electrophore-tograms were obtained at 550nm, using an ISC0 gel scanner accessory in combination with an ISC0 Type 6 optical unit and Type UA-5 absorbance monitor. 

Size-based analysis by CE provides similar information and comparable limits of detection to analysis by SDS-PAGE with Coomassie blue staining.120 129 The performance of both electrophoretic techniques for the analysis of polypeptides is far superior to size exclusion chromatography. Figure 9.7 shows the separation of SDS-complexed recombinant protein standards by CE. 

Sodium dodecyl sulfate-PAGE (SDS-PAGE) was conducted in a 0.5-mm thick 15% horizontal slab gel (77). Samples were prepared in buffer with dithiothreitol and heated to 100°C for 5 min. The gel was prerun for 3 h at 10°C, pH 8.3, and 150 V, and then samples were run for 3 h at 250 V. Protein was again visualized with silver or Coomassie Blue stain. 

Coomassie blue stain 0.025% (w/v) Coomassie brilliant blue R-250 (Bio-Rad) in 40% methanol/7% acetic acid (v/v)... 

Stain membrane with Coomassie blue stain for 5 min. 

An additional visualization technique for PVDF membranes is transillumination, described by Reig and Klein (1988). In that technique, the membrane is dried at room temperature, then wet with 20% methanol and viewed on a white light box. Protein bands appear as clear areas. Sensitivity is usually comparable to that of Coomassie blue staining. 

Fig. 17.1. Parasites cannot hide their spots Two-dimensional electrophoresis gel of the cytosolic fractions of (A) ovine F. hepatica and (B) of a free-living close relative P. nigra run on 17 cm IPG 3-10 and 12.5% SDS-polyacrylamide gels, Coomassie blue stained. The ability to run comparable 2DE arrays will highlight parasite-specific proteins essential to the parasitic lifestyle (Morphew, 2004, unpublished).

Fig. 17.3. The infected bile-ome Two-dimensional electrophoresis gel of F. hepatica infected host bile. Run on a 17 cm, pH 3-10, IPG strip in the first dimension and then on 12.5% SDS-PAGE gel in the second dimension with Coomassie blue staining. Host and parasite proteins were identified via their peptide mass fingerprints (Morphew, 2004, unpublished). Valid parasite secreted proteins can only come from in vivo proteomics.

Fig. 1. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of expression and purification of recombinant protein. Ten-microliter aliquots were withdrawn at each step of the purification and loaded on a 12% SDS-PAGE gel in a Mini Protean III cell gel electrophoresis unit (Bio-Rad). The detection was performed with Coomassie blue staining. MW, low range (14-98 kDa) molecular weight marker (Bio-Rad).

Dissolve the pellet with 5 pL of water. Identify both the supernatant and pellet fractions or both the total and the supernatant fractions by Coomassie blue staining of a 15-25% gradient SDS-PAGE gel (see Fig. 5). Verify that yeast ubiquitin is synthesized and is in the supernatant fraction. 

Stocks are analyzed by silver or Coomassie blue staining of capsid proteins separated on SDS polyacrylamide gels. As shown in Fig. 2.4A, the three capsid proteins (VP1, 2, and 3) are visible in the correct stoichiometry of 1 1 10, and are free of non-AAV proteins (>95% pure). 

DZA and 35S-Met are fluorograms while Protein is Coomassie blue stain. 

Figure 17.2 Purification of M. brassicae male antennal proteins by reverse phase-HPLC. Coomassie blue staining of resulting fractions (T = total antennal extract, 100 antennae). Rectangles indicate the bands submitted to N-terminal sequencing. Each fraction = 200 antennae-equivalent.

Fig. 4.4. Photoaffinity labeling of a benzodiazepine receptor. SDS-polyacrylamide gel electrophoresis of purified synaptic membranes after photoaffinity labeling with [3H]fluni-trazepam (3 nM). Right distribution of radioactivity in the gel. The hatched area is the label distribution when diazepam (10 pM) was present during photolysis (non-specific labeling). Left Coomassie blue staining pattern after irradiation (with and without diazepam present).

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