Amido Black

B destaining solution 10% acetic acid (v/v), 30% methanol or nondenaturated ethanol or isopropanol (v/v) in deionized water. 

Gels may he fixed in Soln. B, i.e., a previous fixation by TCA or 5-sulfosalicylic acid is mostly not necessary. 

Agitate the gel during staining and destaining. Elevated temperature (40 - 50 °C) reduces staining and destaining times considerably. 

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Ami dinoaziri dines 2-Amido Amido black Amidochlor... 

Amido black is a commonly used stain, but it is not very sensitive. It is often used to visualize concentrated proteins or components that are readily accessible to dyes such as proteins that have been transferred from a gel to nitrocellulose paper. Two of the more sensitive and more frequently used stains are Coomassie Brilliant Blue (R250 and G250) and silver stains. Because these stains interact differently with a variety of protein molecules, optimization of the fixative and staining solutions is necessary. The Coomassie stains are approximately five times more sensitive than amido black and are appropriate for both agarose and polyacrylamide gels. The silver stain is approximately 100 times more sensitive than Coomassie and is typically used for polyacrylamide gels. 

To quantitate proteins from staining, a densitometer aided by computer software is used to evaluate band areas of samples compared to band areas of a standard curve. Amido black, Coomassie Brilliant Blue, and silver stains are all appHcable for use in quantification of proteins. 

Orange G, Amido Black 10B, Direct Red 4BS and Congo Red Four bacterial strains (pseudomonads) isolated from dyeing effluent-contaminated soils Maximum degradation observed in the treatment system after 24 h for Orange G was 60.9 mg L 1, for Amido Black 10B 571.3 mg L, for Direct Red 4BS 112.5 mg L, and for Congo Red 134.9 mg L 1 [184]... 

Lentinula edodes MnP Congo Red, Trypan Blue, Amido Black [13]... 

Whereas gels are mainly electroblotted immediately after electrophoresis, it is possible to blot Coomassie or Amido Black stained gel too, but with lower efficiency and after soaking in ddH20 for 15 min and an equilibration step in transfer buffer C (see below) for 30 min. 

Spin the samples with 200 000 x g at 4 °C for 15 -17 h. After the run, displace the gradient using a more dense solution, e.g., 40% sucrose in Soln. A, colored by a droplet Amido Black 10 B solution. The principle of a displacement apparatus is shown in Fig. 5.4. The RNA content of the fractions is measured either by reading the UV absorption at 260 nm or, if labeled material was used, by counting the radioactivity. To monitor the sucrose gradient, estimate the refractive index of the obtained fractions (concentration, density and refractive index of sucrose solutions are given in Table 8.17). 

Amido black dye Two years later, Grassman and Hanning developed another organic dye to be used on filter paper after electrophoresis amido black stain. It has moderate sensitivity. Today, amido black dye is used for colorimetric determination of electroblotted proteins on PVDF (poly-vinylidene difluoride) and nitrocellulose membranes. 

After transfer, stain the membrane with amido black solution for 20—30 min or with Ponceau S for 5 min (see Notes 4 and 5). 

Depending on the supplier and batch of amido black, the sensitivity of protein detection with this reagent may vary If the sensitivity of staining is not satisfactory, it is advisable to dilute the amido black solution 5-10 times with water rather than to increase its concentration. In our hands, amido black purchased from Research Organics (Cleveland, OH) or Sigma Chemical Co. (St Louis, MO) gives optimum results. 

The sensitivity of staining with amido black in water or with Ponceau S is not very high (about 0.5-5 pg depending on protein species). The advantage of these stains is their reversibility... 

Fig. 1. SDS-PAGE pattern of human IgG heavy chain eluted from PVDF membrane. Human IgG (10 pg) was resolved by SDS-PAGE and transferred to a PVDF membrane. The proteins on the membrane were stained with amido black in water, and the heavy chain was excised and eluted with guanidinium hydrochloride/lysophosphatidylcholine. After precipitation with absolute alcohol, the glycoprotein was subjected to analytical SDS-PAGE, and the gel was stained with Coomassie brilliant blue to ascertain its purity. Lanes 1 and 4 original commercial preparation of human IgG lanes 2 and 3 IgG heavy chain eluted from the PVDF membrane lane 5 mixture of molecular mass standards, from top to bottom phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 ldDa), ct-lactalbumin (14.4 kDa).

Acrylamide (20%) with 0.5% bisacrylamide is appropriate for the separating gel. PVDF blots are stained with amido black, and all clearly visible bands are excised for direct microsequencing on the membrane, using an Applied Biosystems 476A or 494 sequencer (37)... 

Alternative staining procedures (reviewed in refs. 1—3) utilize Coomassie blue, Ponceau S, Amido black, India drawing ink, colloidal gold, or silver. A highly sensitive technique utilizing eosin Y has also been described (18)... 

Stain the membrane with amido black or Ponceau S (unitb3.j) to detect proteins. Stain the gel (unitb3.i) after transfer as appropriate to judge transfer efficiency. 

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. 

Amido black 10B stain 0.1% (w/v) amido black (naphthol blue black 10B, Sigma) in 10% (v/v) acetic acid 5% (v/v) acetic acid Plastic boxes... 

Stain membrane with amido black 10B stain for 1 min. 

The protocols for staining with amido black, Coomassie blue, Ponceau S, and AuroDye follow the suppliers recommendations. It should be noted that when staining PVDF membranes with Coomassie blue before N-terminal sequencing, omitting acetic acid from both the stain and destain solution is recommended to minimize potential extraction of protein from the membrane (Speicher, 1989). 

Alkaline hydrolysis, see Saponification Alkaline phosphatase (AP), in immunoblotting, 207-217 Amido black, staining of proteins blots, 199-200 Amino acids... 

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