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 JoVE Biology

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

1, 1

1Protein Expression and Purification Core Facility, EMBL Heidelberg

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    Summary

    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.

    Date Published: 8/14/2009, Issue 30; doi: 10.3791/1350

    Cite this Article

    Lawrence, A., Besir, H. Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid. J. Vis. Exp. (30), e1350, doi:10.3791/1350 (2009).

    Abstract

    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.

    Protocol

    Part 1: Preparation of the CBB staining solution

    1. 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.
    2. 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.

    Part 2: SDS-PAGE

    1. 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.
    2. 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.
    3. Protein samples are loaded on the gel and electrophoresis run for 50 min at 220V.

    Part 3: Staining of the gel

    1. The gel cassette is disassembled and the gel placed in a box for the subsequent washing steps.
    2. 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.
    3. 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.
    4. 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.
    5. The gel can be scanned, photographed or dried for long-term storage

    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.


    Fig. 1: Representative gel stained after loading samples of a protein purification (*: molecular weight marker).


    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).

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    Discussion

    • The washing steps are critical for the efficient staining of the proteins. A reduced washing time below 2 min or reduced volume of water (<50ml) can result in pale blue protein bands, most likely due to higher amounts of residual SDS in the gel.
    • 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 results in crosslinking of proteins to the gel matrix and thus detection of the proteins by mass spectrometry could be hampered.
    • Instead of CBB G-250, one can use CBB R-250 according to the original protocol by Wondrak. We have not compared both dyes side by side as we had a stock of CBB G-250 in our lab and had good results with this dye.
    • The speed of the procedure and the omission of toxic or hazardous solvents in the washing and staining steps are the most important and convincing factors for using this protocol. The sensitivity is in the same range of classical CBB staining protocols and commercial CBB staining solutions and has not been a limiting factor for our SDS-PAGE analysis in the field of expression and purification of recombinant proteins.

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    Disclosures

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

    Acknowledgements

    We would like to acknowledge the technical assistance of Ines Racké.

    Materials

    Name Company Catalog Number Comments
    Coomassie Brilliant Blue G-250 AppliChem A3480 any other CBB G-250 could be used as well
    Concentrated HCl

    References

    1. Process for fast visualization of protein. US patent. Wondrak, E. M. 6319720 (B1) (2001).
    2. Solution for fast visualization of protein. US patent. Wondrak, E. M. 2001046709 (A1) (2001).

    Comments

    11 Comments

    Hi,
    This is very clear and easy way of staining the gels. I would like to know the consequences of using acetic acid and organic solvents in staining the proteins electrophoresd using polyacrylamide gels, or what was is this method advantageous than the traditional coomassie staining using acetic acid and methanol ?
    your replies are appreciated

    regards,
    Rajesh
    Reply

    Posted by: AnonymousAugust 17, 2009, 2:23 AM

    Hi,
    as indicated in the article, the main factors for using this method are avoiding organic solvents and acetic acid for safety reasons and the speed of staining. This method has a lower sensitivity compared to longer protocols but for our applications the speed of staining compensates that.
    We haven't analyzed the effect of using organic solvents or acetic acid versus HCl in water, so I can't tell you if e.g. the fixation of the proteins in the gel is affected or more protein is dissolved from the gel leading to lower sensitivity. I hope this answers your questions.
    Best regards
    Hüseyin
    Reply

    Posted by: Hüseyin B.August 25, 2009, 8:14 AM

    Hi,
    May we have a pdf version of your method ?
    Also, at what temperature the solutions can be heated ?
    Is your method compatible with mass spectrometry-based micriosequencing ?

    Best regards,

    Hristo Atanassov
    Reply

    Posted by: AnonymousNovember 12, 2009, 10:32 AM

    Hi,
    there should be a link to the pdf-file on the page of the video. If you cant find it, please contact me at www.embl.de/services/core_facilities/pepcore/members/index.php?s_personId=4403.
    As described, we heat the gel in the microwave oven for about 10 sec so it's getting hot without boiling. I guess it's about 70-80°C.
    As you may know, you should not heat a gel in the microwave if you want to do mass spec. afterwards as you will decrease the efficiency of extraction of protein bands from the gel. Additionally, the sensitivity of this fast method is lower than for some Coomassie protocols optimized for high sensitivity so it may not be ideal for very low protein amounts (
    Reply

    Posted by: Hüseyin B.November 13, 2009, 4:16 AM

    Hi,
    there should be a link to the pdf-file on the page of the video. If you cant download it, please contact me at www.embl.de/services/core_facilities/pepcore/members/index.php?s_personId=4403.
    As described, we heat the gel in the microwave oven for about 10 sec so it's getting hot without boiling. I guess it's about 70-80°C.
    As you may know, you should not heat a gel in the microwave if you want to do mass spec. afterwards as you will decrease the efficiency of extraction of protein bands from the gel. Additionally, the sensitivity of this fast method is lower than for some Coomassie protocols optimized for high sensitivity so it may not be ideal for very low protein amounts (
    Reply

    Posted by: AnonymousNovember 16, 2009, 12:17 PM

    Hi,
    Thank you for this method. However, I would like to know is there some special procedure when using this method with Protein Agarose gel electrophoresis? I tried with Agarose gel but it appears the entire gel is stained permanently blue. Have you tried this method with agarose gel or modified it for such purpose?
    Thank you.
    Reply

    Posted by: AnonymousSeptember 28, 2011, 8:48 AM

    Hi,
    we haven't tried this with agarose gels, but I'm sure you need to do some modification of the procedure. The most important one would be avoiding the microwave step for washing and staining. This would obviously damage the agarose gel. As far as I know the destaining of agarose gels can take much longer compared to poly-acrylamide gels. Not sure if due to larger pore size thus more dye inside the gel or higher affinity of the dye to agarose due to more polar structure of agarose, maybe both. I hope that helps.
    Reply

    Posted by: AnonymousSeptember 28, 2011, 10:20 AM

    Hi,
    I just want to say how I'm thankful because of sharing this very wonderful fast method by this site. It helped me to have more clear gels and to save the time.
    Thank you again!
    Hanie
    Reply

    Posted by: Hanie k.January 27, 2012, 12:46 PM

    It seems the protocol is simple and easy. I will try. If I want to make just 100mL of staining solution, I would take 6mg dye, 100mL water and 0.3mL HCL. DŒs this work?
    Reply

    Posted by: AnonymousFebruary 13, 2012, 12:34 AM

    Hi,
    I can say, It works, especially when your protein concentration on gel lanes is high enough (according to my own experience). Give it a try!
    Be lucky
    Reply

    Posted by: Hanie k.February 14, 2012, 5:14 AM

    We are using this method but have encountered an occasional glitch... our gels are blue with negative (transparent) bands where the protein should be.... We are considering the possibility that this is the result of insufficient washing prior to staining, but this is not consistent with the gel that you have shown (which was not washed enough).

    Any suggestions would be greatly appreciated.
    Reply

    Posted by: AnonymousFebruary 20, 2012, 10:01 AM

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