Blotting Blue gels

Serva Blue G binds tightly to nitrocellulose and PVDF membranes. The blot of a blue gel is thus blue, and this interferes with the immunostain or ligand coloring of the blot (see Section 1.6.3). Finally, the run of a blue gel lasts 3 to 6 h, and the bands are blurry. 

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... 

Run gels at constant voltage, 25-30 mA per slab gel, until the bromophenol blue marker dye has readied the bottom of the gel (about 1 hour). When completed, turn off and unplug the electrophoresis apparatus and remove the gel. Carefully cut the gel between lanes 5 and 6 so that you have two identical halves. Cut a notch in the corner of each gel half to indicate proper orientation. The half containing lanes 1-5 will be stained with Coomassie Blue to detect the location of all proteins. The other half (lanes 6-10) will be blotted onto a nitrocellulose membrane to analyze for glycoproteins. 

As outlined above, the gold stain should be matched in sensitivity to the amount of protein loaded on the gel This can be estimated as follows if a strip of the gel from which the blot is to be made can be adequately stained with Coomassie blue, citrate gold will yield a good stain of the blot, whereas AuroDye will heavily overstain it. If the gel contains so little protein that it has to be detected by silver staining, AuroDye and the corresponding method (see Methods in Molecular Biology, Volume 3, Chapter 34) should be used. 

Proteins in the middle two-thirds of the gel usually transfer to high-retention PVDF membranes with an average yield of 50% to 80%. The protein pattern on PVDF membranes stained with amido black or Coomassie blue should closely resemble the pattern on duplicate lanes of the gel stained with Coomassie blue. The staining intensity on the blot should be slightly higher than on the gel because the proteins are concentrated on the surface of the blot rather than distributed throughout the thickness of the gel. Proteins below the dye front usually will not be recovered on the membrane. Proteins in the top 20% of the gel are often incompletely transferred out of the gel. 

Fig. 2. Gradient (4-20%) acrylamide gel of bone extracts. Two hundred micrograms of modem bone extract (M) and 400 /ig of three fossil bone extracts from the Archaic (7000 years b.p.) Windover site were stained with Coomassie Brilliant Blue. Products in the molecular weight range of albumin, IgG (heavy chain), and osteonectin are annotated, and the three proteins were identified in Western blots of this gel in both the modem and fossil bone extracts.

Figure 21. A. Reaction of Ca2+-ATPase protein to anti-PHB IgG. Purified Ca2+-ATPase protein (8.5 pg per lane) was separated by electrophoresis on a 10% polyacrylamide gel.30 Left lane 1 Coomassie blue stain of Ca2+-ATPase protein. Left lane 2 Western blot of Ca2+-ATPase protein probed with rabbit anti-PHB IgG. Second antibody was anti-rabbit IgG conjugated to alkaline phosphatase. Color development was with the alkaline phosphatase substrate kit (Bio-Rad). B. Phosphorylation of the Ca2+-ATPase by [32PjpolyP. Purified Ca2+-ATPase protein (2 jig) was phosphorylated at room temperature by [32P]polyP and separated by electrophoresis on a 10% polyacrylamide gel.30 Right lane 1 Coomassie blue stain of phosphorylated Ca2+-ATPase. Right lane 2 Autoradiogram of phosphorylated Ca2+-ATPase.

All sample proteins were run on either 12% or 14% pre-cast Novex 1.0 mm, 10 well gels under non-reducing conditions according to Laemmli (7). Samples were immediately electroblotted to Immobilon-P PVDF membrane using a semi-dry (MilliBlot-SDE) electroblotter essentially quantitiatively (8). After blotting, PVDF was washed briefly in HPLC water and stained with 0.05% Brilliant Blue-G Coomassie (BB-G) /20% methanol /0.5% acetic acid or Amido Black (2). The membrane was kept wet and not allowed to dry (9). Enzymatic digestion was performed as described (2) with all digestion and extraction buffer volumes reduced to 25 pL. 

Carbonic anhydrase and transferrin (60 picomole each) were run on a 10-20% SDS-polyacrylamide Tris-Tricine gel (1) and then electrically transferred to PVDF membrane in CAPS buffer (2). Tlie blotted proteins were stained with Coomassie Blue G-250 and the stained bands were excised for further study. 

Extracts of the R and S biotypes of Eleusine were fractionated by stepwise increases in polyethylene glycol concentration and the various fractions were monitored for the presence of tubulin and other proteins by electrophoresis and Western blotting. By making small increases in PEG concentration, one fraction contained virtually all the recognizable tubulin and was > 85% pure as determined by Coomassie blue staining of denaturing gels (23, 24). This is comparable in purity to the protocols of Morejohn gi al- (25), but is a much faster method and allows... 

Transgenic rice grains carrying the ITF gene were analyzed by Coomassie blue-stained gel and Western blot analysis (Fig. 

Fluorography (see Table 1.3) solves both problems. In case 1, the experimenter soaks the gel with a scintillator liquid (Enhance, Enlightning, Entensify). This converts the P-radiation into light. The light is not absorbed in the gel and thus reaches the film. However, the gels should not be stained with Coomassie Blue because the stain partially absorbs the light. Fluorography is also applicable to nitrocellulose blots. You spray the blot with Enhance Spray by NEN. 

Protein detection Protein quantitation in solution in gels, in CE and 2DE, and on blots SYTO, SYBR, fluorescamine and o-phthaldialdehyde, novel dyes BODIPY, NanoOrange, CBQCA and SYPRO protein gel stains In addition to the conventional dyes for protein staining (Coomassie Blue, colloidal gold), many novel fluorescent stains have been developed that allow highly sensitive protein quantitation in solution and in gels, particularly the SYPRO protein gel stains. 36... 

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