xref Add 100 mL of acetic acid and adjust the total volume to 1000 mL with water. At the end of this time, remove the solution by decanting. Cornell University 0000000000 65535 f /Im0 Do 1 0 obj This is especially important for low level digests. /Font<< The final concentration of acetic acid is 5% (v/v). In this protocol, background staining is low due to the very low dye concentration used. /Root 2 0 R Add 100 mL of acetic acid and, after mixing, adjust the total volume to 1000mL with water. /Type /Page ] Creating an Account to Access BRC Services, Cornell Institute of Biotechnology It might be convenient to carefully transfer the gel to a bag for longer-term storage. /Resources<< /Parent 3 0 R The gel should be exposed to 10% acetic acid, 50% methanol for a total (stain plus destain) period of at least 3 hours (with shaking and at least three solvent changes) to ensure adequate removal of SDS. /Type /Catalog << /Type /Pages q Testing is still in progress to determine if this treatment can be used effectively in humans. Protocols for Staining Gels with Coomassie Blue R-250 or Coomassie Blue G-250. 0000003099 00000 n c;pSirD.qr`*! 0000001965 00000 n 6301 endstream endobj 30 0 obj <>stream The gels are soaked in dye and excess stain is then eluted with a solvent (destaining). >> Destain until background is clear. endobj Continue the destaining until the protein bands are seen without background staining of the gel. This treatment allows the visualization of protein bands. /CreationDate (D:20050721115044) The final concentrations are 50% (v/v) ethanol in water with 10% (v/v) aceticacid. Cool and add 100 ml 2N H2S04. Although G-250 is more sensitive, R-250 affords better resolution, and is often used instead. Cover the gel with gel-washing solution, and continue to fix the proteins in the gel by incubating overnight at room temperature with gentle agitation. Storage solution: Add 25mL of acetic acid to 400mL of water. To filtered solution, CAREFULLY add 22.2 ml 10N KOH, then add 28.7g TCA. 0000004515 00000 n UiN6BEWlIvRCx&jrr;lILT5zL\C2M!M >> << If you have a disability and are having trouble accessing information on this website or need materials in an alternate format, contact web-accessibility@cornell.edu for assistance. %PDF-1.4 Academic press Inc. Peng, Weiguo; Maria L. Cotrina, Xiaoning Han, Hongmei Yu, Lane Bekar, Livnat Blum, Takahiro Takano, Guo-Feng Tian, Steven A. Goldman, Maiken Nedergaard (2009-07-28). /Filter /FlateDecode The absorbance data can then be used in Beers law to determine protein concentration and ultimately the actual amount of protein in a given solution. You may reuse the stain so pour it into a new vial. The recent tests have administered the dye within 15 minutes of injury, but to be effective in a real-life setting, where it may take time for a patient to reach the emergency room, the treatment should be effective even when administered up to two hours after injury. Allow staining to proceed until desired band intensity is reached. If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear. /ID[<0e52fe6aa7d81135b50d2b95372386a8><0e52fe6aa7d81135b50d2b95372386a8>] Destain: Add 500mL of HPLC- grade methanol to 300 mL of water. After electrophoresis, fix gel in 40% methanol/ 50%water/ 10% acetic acid for approximately hr. 32 0 obj <>stream The gel should be covered during this process to avoid contamination and to prevent the evaporation of the solution. /ModDate (D:20050721115044) Cover the gel with the destain solution and allow the gel to destain with gentle agitation. Destain gel in 10% Acetic Acid for 2 hours or more. Cover the gel the Coomassie stain. hbbd`b`eb`Heb`6 ' Gel-washing solution: Add 500mL of HPLC-grade methanol to 300 mL of water. << "aR QA@Izc~tz>h1C-Aaov0)z1 s\7K}eB`hrr}(t/ l0Epzy.=:)_P\L94 EpSQPU6L0;g1nkJcGDnw>!, 0?>Q^ G@Y{ |vhr2P startxref Ithaca, NY 14853 Q Stain the gel with 0.1% (or less) Coomassie Blue R250 in 10% acetic acid, 50% methanol, and 40% H2O for the minimum time (typically less than one hour) necessary to visualize the bands of interest. /Creator >> endobj Stain gel in 10% Acetic Acid in water, containing 60 mg/L of Coomassie Blue R-250. >> Filter the solution to remove any insoluble material. v w >> The leuco form recovers its blue color upon binding to protein, apparently due to a neutral pH environment surrounding the the protein molecule. /Info 1 0 R Email us. Stain gel in the above solution, with 0.25% Coomassie Blue R-250, for 2 4 hours, until the gel is a uniform blue color. 0 %PDF-1.6 % hk..3.~ocb=E)-*yk_)k*xh&\ybB6Ld'r}yi{O^|>pTp{i()kuxq;FNT@s`&0'{N3Hd/# /Producer jsy@S,n7j6{M;Xlz3IkUMw_]JiWd]68^WDL]Uk4Sad 4 0 obj endobj 0000000548 00000 n In addition to their use in gels, Coomassie dyes are an integral component of the Bradford Method for determining protein concentration in a solution. When Coomassie Brilliant Blue G-250 binds to proteins in acid solution, it has an absorbance shift from 465 nm to 595 nm. Coomassie R-250, is the most commonly used variant for detection of protein, allowing for detection of as little as 0.1 ug protein. << Stain the gel at room temperature for 3 to 4 hr with gentle agitation. All Rights Reserved. Without the Coomassie, the technique is known as CN-PAGE (colorless native), and will only separate negatively-charged proteins. ^QJ1]c_$I-w6&Ig9,'yU)#!K Bands will begin to appear within 15 minutes. &. 0 13 The coomassie G-250 dyes is less sensitive, with a lower limit of around 0.5 g for most proteins, but this is somewhat offset by the speed of destaining offered by this dye compared to R-250. NB NB C p p p p p i    , a p      p p #  | p p  : , 8 Alternatively, Coomassie may be added to undenatured protein in PAGE in place of SDS, in a technique called BN-PAGE; both SDS and CBB have the effect of imparting a net negative charge upon the proteins. 0000006226 00000 n hb```f``a '| a0p4a !!A!ax H3``H310500~bx` z Intensity and sensitivity will continue to improve for several hours. Prior to complete staining, the gel will appear as a lighter area against the dark staining solution. << endobj %%EOF. If the protein is not fixed in the gel as a separate step from the staining, the protein will be washed away and your results will be compromised. [1], Brilliant Blue G (BBG) has recently been used in scientific experiments to treat spinal injuries in laboratory rats. After mixing, adjust the final volume to 500mL with water. To stain, immerse gel in above solution. endobj Staining solution is stable for 2 3 weeks @ 25C. endstream endobj 27 0 obj <> endobj 28 0 obj <>>>/Rotate 0/Type/Page>> endobj 29 0 obj <>stream 9 0 obj Coomassie dyes (also known as Coomassie Brilliant Dyes) are a family of dyes commonly used to stain proteins in sodium dodecyl sulfate and blue native polyacrylamide gel electrophoresis (SDS-PAGE and BN-PAGE, respectively) gels. 26 0 obj <> endobj 612.2400055 0 0 789.3600006 0 0 cm xXKo8Y"C8rCJ$m(Kf>O..gf1Foy%eE6pg *h]dl5'E`E&SIwY.nx~72%S!kxzIw;{7y3\|\|wcBrHe%0p!VkG)V -K=o8?G *bk 5 0 obj 1137 endobj /Kids[ Store gels in 7% HoAC. /ProcSet [/PDF /Text] [2] It acts by reducing the bodys natural swelling response, which can cause neurons in the area to die of metabolic stress. endstream Incubate at room temperature 3 hours to overnight, then filter. /Contents 4 0 R >> Stain: Dissolve 0.4g of Coomassie blue R350 in 200 mL of 40% (v/v) methanol in water with stirring as needed. 31 0 obj <>/Filter/FlateDecode/ID[<9CF47CB94E748C41B40F1B5862812B9B><53EF4DBDF158FA4C95BEA70DA499299A>]/Index[26 7]/Info 25 0 R/Length 36/Prev 127568/Root 27 0 R/Size 33/Type/XRef/W[1 2 0]>>stream endstream endobj startxref The gel usually contains a set of molecular weight marker (proteins of pre-determined weight) so that protein molecular weight can be estimated in an unknown solution during the visualization. Store the gel in the storage solution as needed. 9 0 R Staining is complete when the gel is no longer visible in the dye solution. 0000003401 00000 n 0000001767 00000 n ), After electrophoresis, remove the gel from the gel box and plates using a gel separator, Place it in a clean plastic container and cover with ultrapure water, Microwave the gel on high for 1 minute until the solution almost boils (do not overheat), Shake the gel on a shaker for 2 minute and discard the water, After the last wash, add 30 ml of SimplyBlue Coomassie protein stain and microwave on high for 45 seconds to 1 minute until the solution almost boils, Wash the gel in 100 ml of ultrapure water for 10 minutes on a shaker, Add 20 ml of 20% NaCl for at least 5 minutes, Examine gel on a white paper towel for blue-stained protein bands, Discard the gel in the Biohazard waste bin. Allow to stand > 3 hours, then filter again if necessary to obtain an amber-brown solution without blue precipitate. The final concentrations are 50% (v/v) methanol in water with 10% (v/v) acetic acid. trailer Destain for 4 24 hours in 5% MeOH, 7.5% HoAC, 87.5% H2O. 0000005896 00000 n Coommassie Blue G-250 (G for greenish) and Coomassie Violet R-150 later followed. The Coomassie stain is removed by decanting. At the end of this time, remove the solution by decanting. https://openwetware.org/mediawiki/index.php?title=Jacobs:Protocol_Protein_Gel_Staining_Using_Coomassie_Blue_Protein_Stain&oldid=212255, SimplyBlue Coomassie protein stain, 1x pre-mixed solution (Coomassie blue dye binds to proteins (arginine, the aromatic amino acids, and histidine) allowing protein bands to be visualized in polyacrylamide gels. 9 0 i B R p $       i          6 : Coomassie Stain Protocol The gel must be fixed prior to staining by a non-modifying, precipitation procedure such as the ethanol (or methanol)-acetic acid method used here. The final concentrations are 50% (v/v) methanol in water with 10% (v/v) acetic acid. /Size 13 Note: To get the highest sensitivity mix water, MeOH, and Stainer A together; expose the gel in this solution for 10 min; then add the appropriate volume of Stainer B. Cornell Visualization and Imaging Partnership, In-solution digestion for shotgun analysis, Ask Us Anything About Your Needs or Projects. t u v w # To make the Coomassie Blue G-250 staining reagent, dissolve 0.2g dye in 100 ml H2O (this will require warming to approximately 50C). << 4)XNKrGI$H |Wq\]%gs+7rK9jj*("mv9E@rM8|M4)pOHsSL &[sz'U)/Ad,70q4wl2.H7K;,xw-EU;bYViXLFCWzDf }'DEJ;_b~,{1jp,\X:EUpy>BxJQFM1a$L{>gSTp7-1F8#}XpO~R@P:9Uue((qw.XMf%^+j9giA.m~ P.a^-|h2xG{*Vp+j.L/zd gB%`1N\1WpM DmYcKO7j. 0000001788 00000 n Procedure After electrophoresis, the apparatus is disassembled and the gel is washed off the glass plates gel-fixing solution and soaked in that solution for 10 min to 1hr. The original Coomassie dye was developed as a wool dye and named to commemorate the 1896 British occupation of Coommassie (now Kumasi) in Ghana. An Overview of Antigen Retrieval Methods | Protocols Online: [] Tris-EDTA [] An Overview of Antigen Retrieval Methods | Protocols Online: [] Citrate-EDTA Buffer [] Tris Buffered Saline (TBS) Antigen Retrieval Protocol | Protocols Online: [] Buffered Saline (TBS)antigen retrievalso Tris-EDTA Antigen Retrieval Protocol | Protocols Online: [] Tris-EDTA bufferantigen retrievalsolutio An Overview of Antigen Retrieval Methods | Protocols Online: [] EDTA [] Gel may be prefixed in 50% MeOH, 10% HoAC, 40% H2O for 30 minutes to overnight. Bands will begin to appear in 1 2 hours. /Length 5 0 R /MediaBox[0 0 595 842] 2 0 obj Protocols Online 2016. The first of the Coomassie series was Coommassie Blue R-250 (R standing for reddish and 250 being the dye strength indicator). One-Dimensional SDS Gel Electrophoresis of Proteins (western blotting), Preparation of Lysogeny broth (LB) agar plates, Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury, http://www.pnas.org/content/106/30/12489.abstract, http://www.telegraph.co.uk/science/science-news/5921266/Blue-MandMs-mend-spinal-injuries.html, Blue Food Dye Treats Spine Injury in Rats | Wired Science, http://www.wired.com/wiredscience/2009/07/bluerats/, Making stock acid and base solutions (molarities and specific gravities of concentrated acids and bases), DAB-Peroxidase Substrate Solution (Brown). Add 100mL of acetic acid and adjust the total volume to 1000 mL with water. F8 mdZ p#Rf^bVU Ad"S_?v>@ +h*M{7 \?7fiQ%*xKvV+UGt'AW!0|"( /Pages 3 0 R # Published: 7:00AM BST 28 Jul 2009 (2009-07-28). The final concentration is 0.1% (w/v) Coomassie blue R350, 20% (v/v) methanol, and 10% (v/v) acetic acid. /PageMode /UseNone >> L h " B 4 m ( 2 8 A wwwwwwwwwwww hH OJ QJ ^J h`P hH 5OJ QJ ^J hS,- 5OJ QJ ^J h hH 5OJ QJ ^J h hH OJ QJ ^J hyvz hH OJ QJ ^J hH hH 5OJ QJ ^J h, hH 5OJ QJ ^J h%8| hH CJ aJ )hyvz h}t B*CJ0 OJ QJ ^J aJ0 ph . Consequently, the loss in sensitivity of G-250 can offset by the speed and convenience of the protocol, which saves up to 11 hours versus the most sensitive R-250 procedures. stream Gentle agitation of the dishes at all stages of the procedure will help ensure an even treatment if the gel. 6 0 obj << /Type /Font /Subtype /TrueType /Name /F1 /BaseFont /#54#69#6D#65#73#4E#65#77#52#6F#6D#61#6E#2C#42#6F#6C#64 /Encoding /WinAnsiEncoding /FirstChar 32 /LastChar 255 /Widths [ 250 333 555 500 500 1000 833 278 333 333 500 570 250 333 250 278 500 500 500 500 500 500 500 500 500 500 333 333 570 570 570 500 930 722 667 722 722 667 611 778 778 389 500 778 667 944 722 778 611 778 722 556 667 722 722 1000 722 722 667 333 278 333 581 500 333 500 556 444 556 444 333 500 556 278 333 556 278 833 556 500 556 556 444 389 333 556 500 722 500 500 444 394 220 394 520 501 500 501 333 500 500 1000 500 500 333 1000 556 333 1000 501 667 501 501 333 333 500 500 350 500 1000 333 1000 389 333 722 501 444 722 250 333 500 500 500 500 220 500 333 747 300 500 570 333 747 500 400 549 300 300 333 576 540 250 333 300 330 500 750 750 750 500 722 722 722 722 722 722 1000 722 667 667 667 667 389 389 389 389 722 722 778 778 778 778 778 570 778 722 722 722 722 722 611 556 500 500 500 500 500 500 722 444 444 444 444 444 278 278 278 278 500 556 500 500 500 500 500 549 500 556 556 556 556 500 556 500 ] /FontDescriptor 10 0 R>> endobj 10 0 obj << /Type /FontDescriptor /FontName /#54#69#6D#65#73#4E#65#77#52#6F#6D#61#6E#2C#42#6F#6C#64 /Flags 34 /FontBBox [-250 -216 2558 1000] /StemV 77 /ItalicAngle 0 /CapHeight 891 /Ascent 891 /Descent -216 /StemH 77 /XHeight 445 /Leading 149 /AvgWidth 427 /MaxWidth 2558 /MissingWidth 2558 >> endobj 7 0 obj << /Type /Font /Subtype /TrueType /Name /F2 /BaseFont /#54#69#6D#65#73#4E#65#77#52#6F#6D#61#6E /Encoding /WinAnsiEncoding /FirstChar 32 /LastChar 255 /Widths [ 250 333 408 500 500 833 778 180 333 333 500 564 250 333 250 278 500 500 500 500 500 500 500 500 500 500 278 278 564 564 564 444 921 722 667 667 722 611 556 722 722 333 389 722 611 889 722 722 556 722 667 556 611 722 722 944 722 722 611 333 278 333 469 500 333 444 500 444 500 444 333 500 500 278 278 500 278 778 500 500 500 500 333 389 278 500 500 722 500 500 444 480 200 480 541 500 500 500 333 500 444 1000 500 500 333 1000 556 333 889 500 611 500 500 333 333 444 444 350 500 1000 333 980 389 333 722 500 444 722 250 333 500 500 500 500 200 500 333 760 276 500 564 333 760 500 400 549 300 300 333 576 453 250 333 300 310 500 750 750 750 444 722 722 722 722 722 722 889 667 611 611 611 611 333 333 333 333 722 722 722 722 722 722 722 564 722 722 722 722 722 722 556 500 444 444 444 444 444 444 667 444 444 444 444 444 278 278 278 278 500 500 500 500 500 500 500 549 500 500 500 500 500 500 500 500 ] /FontDescriptor 11 0 R>> endobj 11 0 obj << /Type /FontDescriptor /FontName /#54#69#6D#65#73#4E#65#77#52#6F#6D#61#6E /Flags 34 /FontBBox [-250 -216 2568 1000] /StemV 77 /ItalicAngle 0 /CapHeight 891 /Ascent 891 /Descent -216 /StemH 77 /XHeight 445 /Leading 149 /AvgWidth 401 /MaxWidth 2568 /MissingWidth 2568 >> endobj 8 0 obj << /Type /Font /Subtype /TrueType /Name /F3 /BaseFont /#41#72#69#61#6C /Encoding /WinAnsiEncoding /FirstChar 32 /LastChar 255 /Widths [ 278 278 355 556 556 889 667 191 333 333 389 584 278 333 278 278 556 556 556 556 556 556 556 556 556 556 278 278 584 584 584 556 1015 667 667 722 722 667 611 778 722 278 500 667 556 833 722 778 667 778 722 667 611 722 667 944 667 667 611 278 278 278 469 556 333 556 556 500 556 556 278 556 556 222 222 500 222 833 556 556 556 556 333 500 278 556 500 722 500 500 500 334 260 334 584 500 556 500 222 556 333 1000 556 556 333 1000 667 333 1000 500 611 500 500 222 222 333 333 350 556 1000 333 1000 500 333 944 500 500 667 278 333 556 556 556 556 260 556 333 737 370 556 584 333 737 552 400 549 333 333 333 576 537 278 333 333 365 556 834 834 834 611 667 667 667 667 667 667 1000 722 667 667 667 667 278 278 278 278 722 722 778 778 778 778 778 584 778 722 722 722 722 667 667 611 556 556 556 556 556 556 889 500 556 556 556 556 278 278 278 278 556 556 556 556 556 556 556 549 611 556 556 556 556 500 556 500 ] /FontDescriptor 12 0 R>> endobj 12 0 obj << /Type /FontDescriptor /FontName /#41#72#69#61#6C /Flags 32 /FontBBox [-250 -212 2665 1000] /StemV 78 /ItalicAngle 0 /CapHeight 905 /Ascent 905 /Descent -212 /StemH 78 /XHeight 452 /Leading 150 /AvgWidth 441 /MaxWidth 2665 /MissingWidth 2665 >> endobj 3 0 obj Reagents (note- you can skip steps 1 and 2) Gel-fixing solution: Add 500mL of USP-grade 95% (v/v) ethanol to 300 mL of water. /F1 6 0 R /F2 7 0 R /F3 8 0 R>> /Count 1 > 0 2 / C bjbj,(,( . 0000000010 00000 n 0000004802 00000 n /Author The bands develop rapidly and there is no need to destain, for the background color is so light as to be essentially clear. Consequently, a gel placed in an acidified solution of Coomassie G-250 will manifest blue protein bands on a light amber background. This page was last edited on 15 June 2008, at 15:25. /Title The most commonly used dyes in the laboratory for staining PAGE gels are Coomassie Blue R-250 and G-250. Add 200mL of 20% (v/v) acetic acid in water. 0 @ &. The purpose of this step is to gently remove the gel from the plate and begin washing the SDS-containing gel buffers out of the gel. The gel should return to its original dimensions during this process. Destain the gel by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. %%EOF Although R-250 is typically color invariant, Coomassie G-250 undergoes a color shift where the dye changes from dark blue black at pH 7 to a light tan below pH 2. 0000006159 00000 n Equilibrate the gel in storage solution for at least 1 hr. Methods in Enzymology volume 182. Merril CR (1990) Gel-staining techniques In Deutscher MP (Ed) Guide to protein purification. The only reported side effect was that the rats turned blue.[3][4]. The Coomassie dyes (R-250 and G-250) are used for quantification of protein, and work by binding to proteins through Van der Waals attractions and through ionic interactions between dye sulfonic acid groups and positive protein amine groups . One may also note that handling of the gel should be minimized and that gloves should be worn at all times. eU4 7&Hax?2u!8>hdulbytbQ{m?!0TzdmXnfl]:h)Qk~[jFoAwI.ehuOhq$Ul The destain solution should be changed several times, removing it at each change by aspiration. Expose the gel in staining solution overnight and destain the gel by changing water frequently. 130 Biotechnology Building Fix gel in 25% IPA, 10% HoAC in water, 30 60 minutes. E 5 B C Bands will appear in 30 minutes. These steps will minimize surface contamination of the gel.