Coomassie blue staining electrophoresis

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comparable 2DE arrays will highlight parasite-specific proteins essential to the parasitic lifestyle (Morphew, 2004, unpublished).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 Host and parasite proteins were identified via their peptide mass fingerprints (Morphew, 2004, unpublished).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 Ten-microliter aliquots were withdrawn at each step of the purification and loaded on a 12% SDS-PAGE gel in a Mini Protean III cell gel electrophoresis unit (Bio-Rad).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).

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).

Figure 6-4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of normal red cell membrane proteins with Coomassie blue staining.

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.

Silver staining of proteins after their separation by electrophoresis is based on binding of silver ions to sulfhydryl and carboxyl groups of proteins. The proteins are detected as black precipitate of silver. The sensitivity of this method is 20-100 times more sensitive than Coomassie blue staining. 

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... 

At present two-dimensional polyacrylamide gel electrophoresis in one of the most advanced electrophoretic procedures the main obstacle with this method is a reliable interpretation of very complex electrophoretograms containing several hundreds of zones. The procedures for computer evaluation of such maps are available from more specialized literature [117]. Another problem is that the first-dimension run (rod gel) must be done with much less material than the amount required for optimum detection in the slab gel step. With Coomassie Blue staining (and related methods) this is bypassed by overloading the rod gel at the expense of poorer resolution. More sensitive detection methods seem to be the solution here (Fig. 5.15) [118]. 

P]NAD is employed as substrate in this reaction, the ADP-ribosylated protein may be identified by the method of polyacrylamide gel electrophoresis followed by autoradiography of the gel. This method was employed in an examination of the membrane proteins of control and desensitized platelets. Coomassie-blue staining of the gels revealed no differences following desensitization, but there was a > 80% loss of the 45 000 Da protein as revealed by [ P]ADP-ribosylation. The autoradiograph and scans are shown in Figure 8.26. 

Figure 1. Formation of a cross-linked ETF MCAD complex. ETF (9.5pM), MCAD (38 pM) and octanoyl-CoA (l.SmM) were preincubated in 20 mM potassium buffer, pH 8.0 (lane C). ETF (18 pM) and MCAD (18 pM) were also incubated separately with ootanoyl-CoA in buffer (lanes A and B). After 3 mins DMS (9.5 mM) was added and the mixture incubated for 30 mins at 30 °C. After boiling, aliquots were subjected to electrophoresis on an 8% SDS polyacrylamide gel. Densitometric scans of the Coomassie Blue-stained gels are shown.

Thioredoxin f was purified from spinach leaves and analysed by conventional sequencing as described elsewhere [5]. FTR was purified essentially as described earlier [6] substituting a Fd-affinity column for the last two chromatographic steps. The subunits of FTR were separated by SDS-polyacrylamide gel electrophoresis and after Coomassie blue staining extracted and purified according to [7]. The sequences were determined by conventional methods as described elsewhere [8]. 

Fig. 1. Protein patterns of the subnuclear fractions. 12% polyacrylamide gel electrophoresis in 0.1% SDS. Coomassie blue staining. A Nuclei B DNase-released C RNase-released D ammonium sulphate extract nuclear matrix...

To determine which proteins contained (ADP-ribose) , fraction V was analyzed by acetic acid/urea polyacrylamide gel electrophoresis. As shown in Fig. 2A, the major portion of radioactivity migrated with histone HI and radioactivity was also found with staining bands of the four HMG proteins. Following 3-ABm treatment for 16 h, ADP-ribosylation of HMG 14 and 17 was almost completely inhibited, while that of HMG 1 and 2 and histone HI decreased less. The reduced ADP-ribosylation cannot be attributed to differences in protein extraction or proteolysis since Coomassie blue staining patterns were very similar and 3-ABm treatment did not affect the incorporation of labeled lysine into HMG proteins and histone HI (Fig. 2B). ADP-ribosylation... 

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