Coomassie blue staining proteins

Fig. 3A-C. ADP-ribosylation of plasmacytoma free mRNP in presence of RNase A. After incubation of free mRNP with l P] NAD (1 Mci) as described in [4] and variable amounts of RNase A (A control, B 0.5 Mg ml, C 2 Mg ml ) the acid insoluble products were analyzed by 12% polyacrylamide gel containing LDS. S Coomassie blue-stained proteins. Slots 1 to 3 autoradiograms at 1,5 and 20M f NAD...

HS-C protein is poly(ADP-ribose) polymerase. To test this possibility further, the protein was subjected to ADP-ribosylation with [i C]NAD and nicked DNA. The reaction product was subjected to SDS-PAGE. Most of the radioactivity was located near the origin or at a position that approximately corresponded to the Coomassie blue-stained protein band in a parallel track (data not shown). Since poly(ADP-ribose) polymerase is capable of auto-ADP-ribosylation and this modification can alter electrophoretic mobility (6), the two radioactive peaks observed after ADP-ribosylation of HS-C protein probably correspond to the different levels of ADP-ribosylation occurring in the enzyme molecule. That the radioactive peaks were indeed generated by a specific enzymatic reaction was shown by the lack of incorporation of [i C]NAD at 4°C or in the absence of nicked DNA (data not shown). 

Excise with a scalpel the Coomassie blue-stained protein bands carefully, so that only stained (protein containing) membrane slices are used for hydrolysis. 

Fig. 6. Gel electrophoresis of cytochrome oxidase in dodecylsulfate medium. Densitometric tracing of the Coomassie-blue-stained protein bands. ...

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. 

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. 

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. 

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. 

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).

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. 6.3. SDS/PAGE (5-15% linear gradient gel) separated proteins of the total worm homogenate and isolated brush border fractions from protoscoleces of Echinococcus granulosus (horse strain), (a) Coomassie blue staining (b) Periodic acid-Schiff (PAS) staining. (After McManus Barrett, 1985.)...

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.

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