Name: Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid (Video) | JoVE Journal
Uploaded: 2009-08-14T00:00:00.000+00:00
Duration: 7 min 47 s
Description: Learn a simplified protein staining protocol using Coomassie Brilliant Blue G-250 in polyacrylamide gels without organic solvents or acetic acid. This method enables clear protein band visualization after just one minute.

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5 minutes

We would like to acknowledge the technical assistance of Ines Rack&eacute;.

Part 1: Preparation of the CBB staining solution
<ol>
<li>60-80 mg of CBB G-250 are dissolved in 1 liter of bidistilled water by stirring for 2-4 hours. Finally, 3 ml of concentrated HCl is added to the dark blue solution with stirring for another minute and stored in the dark for later use. The solution can be stored for weeks up to several months without losing its staining efficiency.</li>
<li>Concentrated HCl should be handled with the usual care und used under a fume hood. The final solution will be at about pH 2, so gloves should be used and any contact with the skin should be avoided.</li>
</ol>
Part 2: SDS-PAGE
<ol>
<li>Appropriate aliquots of protein samples are mixed with loading buffer to a final concentration of 1x loading buffer. We use 2x loading buffer with 125mM Tris/H3PO4 (pH 7.5 at 25°C), 2mM EDTA, 4% SDS, 200mM DTT, 0.02% bromophenol blue and 50% glycerol. Other loading buffers for SDS-PAGE can be used as well.</li>
<li>Protein samples are heated for about 5 min before loading. Meanwhile, the gel electrophoresis chamber is prepared for the run. We use precast 4-12% NuPAGE© Bis-Tris gels (Invitrogen) in a XCell SureLock® Mini-Cell (Invitrogen) with MES-buffer as the running buffer but any other gel and electrophoresis system can be used as well.</li>
<li>Protein samples are loaded on the gel and electrophoresis run for 50 min at 220V.</li>
</ol>
Part 3: Staining of the gel
<ol>
<li>The gel cassette is disassembled and the gel placed in a box for the subsequent washing steps.</li>
<li>About 100 ml of bidistilled water is added to the gel and heated in the microwave oven for 30 seconds. Heating should be stopped before boiling occurs. The box with the gel is then placed on a shaker for 3-5 min. and this washing step is repeated twice with fresh water.</li>
<li>Enough CBB staining solution is added to cover the gel in the box and the box heated in the microwave for 10 sec. without boiling. The box with the gel is then placed on a shaker for finishing the staining. Already after 1 minute, protein bands can be observed, after 15-30 min. the staining is strong enough in most cases.</li>
<li>The staining solution is poured off and 50-100 ml bidistilled water is added in order to further destain the light blue background of the gel on a shaker. The water can be replaced by fresh water for further destaining if needed.</li>
<li>The gel can be scanned, photographed or dried for long-term storage</li>
</ol>
Part 4: Representative Results:
See Fig. 1 for a properly stained gel following the described procedure.
See Fig. 2 for a gel that has not been washed long enough before staining and residual SDS inhibits efficient staining. Note that the marker lanes (*) contain the same amount of marker proteins.
<img src="/files/ftp_upload/1350/Fig1.png" alt="SDS-PAGE gel electrophoresis; protein separation; molecular weight analysis; laboratory result" data-altupdatedat="1787630408000"> Fig. 1: Representative gel stained after loading samples of a protein purification (*: molecular weight marker).
<img src="/files/ftp_upload/1350/Fig2.png" alt="Electrophoresis gel result, protein bands, molecular weight analysis, laboratory experiment." data-altupdatedat="1787630408000"> Fig. 2: CBB stained gel that has not been washed long enough before CBB staining. The protein bands appear weaker (note that the marker lane * contains the same amount of protein as in Fig. 1).

<ol><li>Process for fast visualization of protein. US patent. , 6319720 (B1) (2001).</li>
<li>Solution for fast visualization of protein. US patent. , 2001046709 (A1) (2001).</li>
</ol>

No conflicting interests. The procedure described above was originally published in a patent application by E.M. Wondrak (see Ref.).

<ul>
<li>The washing steps are critical for the efficient staining of the proteins. A reduced washing time below 2 min or reduced volume of water (&lt;50ml) can result in pale blue protein bands, most likely due to higher amounts of residual SDS in the gel.</li>
<li>If the proteins are going to be analysed by mass spectrometry, the heating steps in the microwave oven should be skipped, the time for washing of the gel extended to about 10 min in each step and the staining time extended until the band intensity is strong enough. Heating the gel...

<table><tbody><tr><td>Coomassie Brilliant Blue G-250</td><td>AppliChem</td><td>A3480</td><td>any other CBB G-250 could be used as well</td></tr><tr><td>Concentrated HCl</td><td></td><td></td><td></td></tr></tbody></table>

staining of proteins in gels with coomassie g-250 without organic solvent and acetic acid

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

To begin this procedure, Kumasi Brilliant Blue or CBG two 50 is dissolved in one liter of bi distilled water by stirring for two to four hours. Hydrochloric acid is later added to the dark blue solution with stirring for another minute, and the solution is stored in the dark for later use. Following protein sample preparation and STS page, the gel cassette is disassembled and the gel placed in a box for three subsequent washing steps with bi distilled water.After the third washing step, the water is replaced with enough CBB staining solution to cover the gel in the box. The box is heated in the microwave for 10 seconds, and the box with the gel is then placed on a shaker. For completion of staining, protein bands can be observed after one minute.Hi, I'm Amri Lawrence from the protein expression purification cough facility at Emberin Heidelberg. Today we'll show you a protocol for staining proteins with cooma brilliant blue G hundred 50 in chloride gels. Unlike classical staining methods, our protocol does not use any organic solvents or acetic acid.We use this protocol to analyze our proteins after every purification step. So let's get started. To prepare the CBB staining solution, 60 to 80 milligrams of CBB is dissolved in one liter of bist distilled water by stirring for two to four hours.After the CBV is dissolved, transfer the solution into a fume hood. In the fume hood, carefully add three milliliters of concentrated hydrochloric acid to the dark blue solution. With stirring for one minute gloves should be worn since the final solution will have a pH of two.Once the solution is made, it is stored in the dark for later use. The solution can be stored for weeks or up to several months without losing its staining efficiency. To prepare protein samples for SDS page, appropriate aliquots of protein samples are mixed with loading buffer to a final concentration of one x loading buffer heat the protein samples for about five minutes before loading.While the samples are being heated, the gel electrophoresis chamber is prepared for the run. We use precast gels in a mini cell with MES buffer as the running buffer. However, any other gel and electrophoresis system can be used.The heated protein samples are then loaded on the gel and electro East for 50 minutes at 220 volts. When the SDS page is complete, the gel cassette is disassembled and the gel is placed in a box with 100 milliliters of bist distilled water. For the subsequent washing steps, the box is heated in the microwave oven for 30 seconds.Heating should be stopped before boiling occurs. After microwaving the box with the gel placed on a shaker for three to five minutes, repeat this washing step twice with fresh water. After the three washes with water, CBB standing solution is added to cover the gel in the box and the box is heated in the microwave for 10 seconds without boiling.After that, the box with the gel is placed on a shaker for completion of the staining. Protein bands can be observed after one minute and after 15 to 30 minutes. The staining is usually strong enough.The staining solution is poured off and 50 to 100 milliliters of bi distilled water is added. In order to further detain the light blue background of the gel on a shaker, the water can be replaced by fresh water for further des staining. If needed, the gel can now be scanned, photographed, or dried for long-term storage.A gel that has been stained following this procedure should look like this. In this gel, the protein bands are stained well and clearly visible. While the background is negligible.The first lane indicated by the asterisk contains molecular weight markers. In contrast, a gel that was not washed sufficiently prior to staining will look like this In this gel, the washing steps were too short, so the protein bands were not stained well enough. Note that the lane indicated by the asterisk contains the same protein amounts of molecular weight markers as a previous gel, but shows less efficient staining.We've just shown you how to stain proteins with co massive brilliant blue in polyamide gels without having to use any toxic or flammable organic solvents. When carrying out this procedure, it's important to remember that the washing steps are critical for the efficient staining of the proteins. So that's it.Thanks for watching and good luck with your experiments.

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Learn a simplified protein staining protocol using Coomassie Brilliant Blue G-250 in polyacrylamide gels without organic solvents or acetic acid. This method enables clear protein band visualization after just one minute.

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