Destaining

Fig. 24 Diffusion destaining apparatus (Desaga). The dish contains the wash liquid and is periodically tilted so that the reagent excess is removed from the chromatogram plate.

Single fusion events of glutamatergic vesicles have been then monitored by following FM 4-64 destaining (Fig. 12.2), a sequence of the stereotyped fluorescence... 

If the stain appears washed out, it is likely that the slide was destained too much. This washed-out appearance can be either because the specimen was left too long in the acid-alcohol destain or because the alcohol wash after the acid-alcohol destain had become acidic as a result of transfer by previous slides. 

Should the stain be unsatisfactory, the slide can be destained by placing it in xylene to remove the cover slip or immersion oil and then placing it in 50% alcohol for 10 min to hydrate the slide. Destain the slide in 10% acetic acid in water for several hours, and then wash it thoroughly first in water and then in 50 and 70% alcohols. Place the slide in stain for 8 min, and then complete the stain procedures. It is helpful to eliminate or shorten the destain step. 

Destain the smear with 1% sulfuric acid for 2 min or until no color runs from the slide. 

Shtilbans V, Szporn AH, Wu M, et al. p63 immunostaining in destained broncho-scopic cytological specimens. Diagn. Cytopathol. 2005 32 198-203. 

Fig. 8.11 (A and B) Expression and purification of insulin-polymer fusion protein detected in copper (A) and Coomassie (B) stained gels. The same gel was first stained with copper, destained and restained with Coomassie R-250. Lane 1 Prestained marker Lane 2 Purified extract of polymer-insulin fusion protein from the chloroplast vector pSBL-OC-40Pris Lane 3 Reverse orientation of fusion protein from pSBL-OC-40Pris Lane 4 Purified extract of polymer-insulin fusion protein from the chloroplast vector pLD-OC-40Pris ...

CBB G-250 is less soluble than the R-250 variety. In acidic solutions it forms colloidal particles that are too large to penetrate surface gel pores and can be formulated into a staining solution that requires little or no destaining. It can also be formulated to be environmentally benign. This stain is somewhat more sensitive than CBB R-250, in part because of increased signal-to-noise ratios... 

The dye Coomassie Brilliant Blue R250 nonspecifically binds to all the protein. The gel is soaked in the dye for it to seep in and bind to the proteins. The gel is then destained to remove the unbound dye. The dye binds to the protein and not the gel, and hence the protein bands can be visually seen. The binding of the dye to the protein is approximately in stoichiometry, so the relative amounts of protein can be determined by densitometry. For most SDS and native gels, separated proteins can be simultaneously fixed and stained in the same solution. 

Destaining Solution Mix 400 ml of Methanol and 70 ml of acetic acid, finally bringing the volume to 1 litre with water... 

Add the destaining solution and shake it slowly. For rapid destaining, microwave on high for 1 min and add some Kim wipes or folded paper towel or cotton. 

Shake it till it destains. One could also replace if the staining solution is too coloured. 

Make the standard curve in the range from 10 to 200 pg BSA. Drop samples and standards onto small sheets of glass filter paper (e.g., Whatman GF/A the sheets are labeled with a pencil). Stain the sheets with Soln. A for 20 min and destain with C until the background is nearly colorless. Extract the sheets with 2.0 ml of Soln. D each after drying and measure the resulting blue solution as described above. 

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

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

TCA, equilibrate the gel twice for 20 min in Soln. B prior to staining. Destaining has to be done alternating with Soln. B and B. ... 

The destained gel maybe stored in Soln. B for several days, but weak bands fade slowly. 

By modification of Soln. A (0.05 g of dye are dissolved in 10 ml glacial acetic acid and filled up with deionized water to 100 ml), staining is done at 60 °C and destaining is done with 10% acetic acid in deionized water. 

A 0.25 g Fast Green are dissolved in 30 ml ethanol and 10 ml glacial acetic and filled up to 100 ml with deionized water B destaining solution 10% acetic acid (v/v), 30% methanol or denaturated ethanol (v/v) in deionized water... 

Acidic fixed gels are equilibrated three times for 20 min in Soln. B. Then the gel is stained with Soln. A for 1 h in the dark followed by destaining in Soln. B. 

Stains All is light-sensitive and bleaches slowly at the air. The stained gel should dry in a vacuum gel dryer immediately after destaining and documentation. 

Staining is done by incubation with a tenfold gel volume of Soln. A overnight. Use several changes of Soln. B for destaining. Remaining colloidal dye on the surface of the gel is wiped off with a wetted tissue. 

The weakening or destaining is performed under visual control and finished by incubation with ddH20. 

Stain the gel for 1 h in Soln. B, pour off the liquid and destain by several changes of Soln. C until the background is colorless. 

A nitrocellulose sheet is stained with Soln. B immediately after electrotransfer or dot blotting. Soln. D is used for destaining. The detection limit is about 20 ng/band. 

For complete destaining the NC sheet is agitated in Soln. F for 15-30 min, followed by rinsing with Soln. G to remove traces of dye. 

The membrane is rinsed with H2O after protein transfer (electrotransfer or dot blot) and dipped to methanol for some seconds. Immediately after this (avoid drying), the membrane is agitated in Soln. A for maximum 2 min and destained by several changes of Soln. B. When the membrane is dry, blue bands are visible on a slight blue background. Membranes stained by this method are not suitable for any immunochemical detection. 

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