Brilliant Blue Staining

The larg staining visualize and mos molecuh addition after tb SDS-cor between concent quantita 

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Alternate Protocol 4 Denaturing Isoelectric Focusing Support Protocol 1 Coomassie Brilliant Blue Staining of Polyacrylamide B3.1.12... 

Adjust sample concentration to 0.5 to 10 mg/ml for Coomassie brilliant blue staining or 0.05 to 1 mg/ml for silver staining. 

COOMASSIE BRILLIANT BLUE STAINING OF POLYACRYLAMIDE GELS... 

Add just enough Coomassie brilliant blue staining solution so that the gel floats freely in the tray. Shake slowly 4 hr to overnight on a laboratory shaker or rocker. 

Additional reagents and equipment for SDS-PAGE (see Basic Protocol 1) and Coomassie brilliant blue staining and drying gel (see Support Protocol 1). 

Coomassie brilliant blue staining (see Support Protocol 1) is based on the binding of the dye Coomassie brilliant blue R250, which... 

With Coomassie brilliant blue staining one should be able to detect -50 to 100 ng protein in a normal band. The lower detection limit of silver staining is -1 ng protein. Once the gel is stained, it can be photographed or dried on a transparent backing for a record of the position and intensity of each band. 

Fig. 7. Activity and folding of the cell-free produced polypeptides. Autophosphorylation activity of fixsArabidopsis protein kinases. Sodium dodecyl sulfide-polyaciy-lamide gel electrophoresis and Coomassie Brilliant Blue-stained gel of the partially purified products marked with asterisks (A), and the autoradiogram (B). Lanes 1-6 represent Atlg07150, At5g49760, At2g02800, At5g62710, and At4g35500, respectively. NC denotes samples from the reaction mixture incubated in the absence of mRNA. M protein size marker. Note that product of Atlg07150 (lane 1) did not show activity.

The sample detection after electrophoresis can be quantified by a densitometric analysis of Coomassie brilliant blue stained spots. A silver stain is available to provide higher sensitivities when detection of samples in the nanogram range is required. 

Another method consists of a spectroscopic measurement of Comassie brilliant blue stained SC protein directly on the tape.17 In contrast with the colorimetric method described earlier, this method does not require any SC extraction procedure prior to protein assay. However, it has been shown to be variable and not appropriate for quantitative determination since the absorbance of colored SC proteins is negligible as compared to light scattering of the SC material adhering to tape strips. 

For identification, protein spots were cut from colloidal Coomassie Brilliant Blue-stained gels equivalent to the gel shown in Fig. 14B. Excised... 

Apoprotein composition was analysed by denaturing SDS-polyacrylamide gel electrophoresis/Coomassie Brilliant Blue staining and densitometry lipid composition (NEFA, TAG, cholesterol and cholesterol ester, and phospholipid) was determined with commercial kits (Sigma). 

Fig, 3. (a) Elution profile of TX-REC when applied to HiTrap Heparin affinity column. The firac-tions containing Tk-REC are indicated, (b) Coomassie Brilliant Blue stained SDS-PAGE of the 10 consecutive fractions containing purified recombinant Tt-REC eluted from the HiTrap Heparin affinity column. Lane M, molecular mass standards lanes 1-10, purified recombinant Tk-REC from fractions 1 to 10, (c) DNase activity of purified recombinant Tk-REC from fractions 1 to 10. Lane C, pUC19 (2 xg) without addition of Tfe-REC lanes 1-10, pUC19 (2 p,g) with Tk-REC (10 p.1) of fractions 1 to 10. Arrows indicate nicked DNA with slow migration rates. 

A Coomassie brilliant blue stained SDS polyacrylamide gel of purified PS II complexes from nidulans. 

Fig. 3. (left) SDS-PAGE of p PJNAD treated SR vesicles in the absence and presence of poly L-lysine. 0.88 mg/ml of SR was incubated at 25 C for 60 min with 20 [xM [32p]NAD (2.8 ci/mmol) and 0, 50 or 100 M,g/ml of poly L-lysine. Radiolabeling of the acid insoluble fraction (25 p.g protein) from each sample was analyzed by SDS-polyacrylamide gel electrophoresis (8). Tlie Coomassie brilliant blue-staining pattern (A) and an autoradiogram of the same gel (B) are shown. Molecular weight markers phosphorylase b (94 K), bovine serum albumin (67 K), ovalbumin (43 K), carbonic anhydrase (30 K) and soybean trypsin inhibitor (20.1 K). 

Fig. 3. Autoradiograph of [ P]ADP-ribosylated proteins in V-79 and variant cells. Cells were penneabilized and 1x10 cells incubated at 37°C for 15 min with 1 nM [ P] NAD (specific activity 7800 cpm/pmol) and 3(X) l nA DNase to induce maximal DNA damage. Reactions were stopped by addition of sample solution and boiled 2 min. Solubilized samples were electrophoresed on a SDS, 9% polyacrylamide slab gel with a 3% polyacrylamide stacking gel and the autoradiographed. Lanes A, B, and C represent V-79, variant ADPRT 54 and variant ADPRT 351, respectively. Autoradiographs were exposed for 24 hr. Molecular weights indicated on the left of autoradiograph were determined by Coomassie Brilliant Blue staining of standards included in the slab gel.

There are many situations in which one would like to recover proteins from acrylamide gels for further analysis. For example, polypeptides extracted from gels can be readily used to immunize rabbits or mice to prepare antibodies (see articles by Christian Huet and by Ariana Celis, Kurt Dejgaard, and Julio E. Celis). Here we present a simple protocol for electroeluting proteins from fixed, unstained and Coomassie brilliant blue-stained dry two-dimensional (2D) gels (lEF or NEPHGE). 

Cut spots from the dry, Coomassie brilliant blue-stained gels using a... 

FIGURE 1 Cutting polypeptides from a dry Coomassie brilliant blue-stained lEF 2D gel of SV40-transformed human keratinocytes. 

Figure 3 A shows a silver-stained gel of electroeluted glutathione S-transferase ti (GST-tt) cut from a dry Coomassie brilliant blue-stained 2D gel of SV40-transformed keratinocytes. The sample was co-run with a small amount of [ S]methionine-...

Coomassie brilliant blue staining solution Dissolve 1.0 g of Coomassie blue in 400 ml of methanol and stir for 1 hr. Add 10 ml of glacial acetic acid and 590 ml of water. Stir for 30 min and filter through a Eluoropore membrane. 

Methods for Visual and Fluorescent Staining of Proteins Resolved on Gels 1. Coomassle Brilliant Blue Staining... 

Kushi and Handa (68) described in 1985 a TLC/MS method for the analysis of lipids. Secondary ion mass spectrometry with a liquid matrix of triethanolamine was used for the extraction and ionization of sample spots first located with iodine or Coomassie brilliant blue staining. A piece of TLC plate of size 5 x 20 mm could be attached to the direct insertion probe, and scanning in one-dimension was accomplished by manually inserting the probe into the source of the mass spectrometer. Spectra could be obtained from one microgram of a lipid separated on a silica TLC plate with aluminum- or plastic-backed TLC plates. Although not specifically noted in this paper, since a plastic-backed plate is an electrical insulator, some provision for connecting the surface to the plate platform itself must be made to hold the surface at the source potential. 

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