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Dimethylmethylene Blue Assay (DMMB)    

Vivien Jane Coulson- ThomasVivien Jane Coulson- Thomas Tarsis Ferreira GesteiraTarsis Ferreira Gesteira
DOI:   10.21769/BioProtoc.1236
Published:   Vol 4, Iss 18, September 20, 2014
Biochemistry > Carbohydrate > Glycoprotein
Biochemistry > Carbohydrate > Polysaccharide
Cell Biology > Cell staining > Carbohydrate
Mammalia > General > Cell line > Physiology
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In this protocol

Original research article

A brief version of this protocol appeared in:
Stem Cells
Oct 2013

 

Abstract

Glycosaminoglycans (GAGs) are long unbranched polysaccharides consisting of repeating disaccharide units composed of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (glucuronic or iduronic acid). Depending on the disaccharide unit the GAGs can be organized into five groups: chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate and hyaluronic acid. The GAGs are heterogeneous molecules with great variability in molecular mass and both sulfation density and pattern. Spectrophotometric assays to measure the GAG content in biological fluids and tissue/cell extracts are valuable tools. The dye 1,9-dimethylmethylene is a thiazine chromotrope agent that presents a change in the absorption spectrum due to the induction of metachromasia when bound to sulfated GAGs enabling rapid detection of GAGs in solution (Whitley et al., 1989; Chandrasekhar et al., 1987; Farndale et al., 1982). Moreover, there is a window in which a linear curve may be drawn (approximately between 0.5-5 μg of GAGs) enabling the quantification of GAGs in solution.

Keywords: Glycosaminoglycan quantification, Proteoglycans, Sulfated Glycosaminoglycan

Materials and Reagents

  1. Dimethylmethylene blue (DMMB) (Sigma-Aldrich, catalog number: 341088 )
  2. NaCl
  3. Glycine (Sigma-Aldrich, catalog number: 410225 )
  4. Glacial acetic acid (Sigma-Aldrich, catalog number: S7653 )
  5. Tris-Base (Merck KGaA, catalog number: 648310 )
  6. Bovine chondroitin 4-sulfate as standard (Sigma-Aldrich, catalog number: C9819 )
  7. DMMB reagent (see Recipes)

Equipment

  1. Plate mixer (VWR International, catalog number: 89202-332 )
  2. Cover adhesive (R&D Systems, catalog number: DY992 )
  3. Microplate reader with 525 nm (BioTek Instruments, catalog number: 11-120-531 )
  4. 96 well microplate spectrophotometer with 525 nm filter set (Thermo Fisher Scientific, catalog number: 51119200 )
  5. Microplate shaker (VWR International, catalog number: 97043-608 )

Procedure

  1. Prepare DMMB reagent and paper filter using Whattman® 3MM. The pH of this solution is around 3.0. To prepare 1 L dye solution, dissolve 16 mg DMMB in 1 L water containing 3.04 g glycine, 1.6 g NaCl and 95 ml of 0.1 M Acetic Acid.
  2. Prepare standard solution of chondroitin 4 sulfate (500 µg/ml in H2O). Prepare standard curve as stated in the table bellow.
  3. Pipet the standard stock solution and complete the volume to 20 µl with H2O into the 96 well microplate.
  4. Pipet 20 µl of each sample into the microplate.
  5. Add 200 µl of DMMB to each sample and shake the plate of a plate shaker for 5 sec.
  6. Read the absorbance using a plate reader at 525 nm immediately.

    Std (µg/ml)
    		 Vol (µl) of 500 µg/ml std
    		 vol H2O (µl)
    		 vol DMMB (µl)
    0
    		 0
    		 20
    		 200
    1.25
    		 2.5
    		 17.5
    		 200
    2.5
    		 5
    		 15
    		 200
    5
    		 10
    		 10
    		 200
    7.5
    		 15
    		 5
    		 200
    10
    		 20
    		 0
    		 200

Notes

  1. DMMB assay can normally be performed on samples with high detergent and salt concentrations; however the standard curve should be prepared in the same solution.
  2. Avoid to performing the assay on samples in high albumin or serum concentrations which may interfere with the assay (Warren, 2000).
  3. Some groups have reported the interference of DNA in the DMMB assay, however, decreasing the pH to approximately 3 and increasing salt concentrations makes the interference of DNA negligible.
  4. DMMB requires the length of glycosaminoglycan chain be over a tetrasaccharide.
  5. DMMB reacts with the sulfate group of the GAG chain and therefore will not work with unsulfated GAGs such as hyaluronic acid.
  6. Further information can be acquired by utlilizing the carbazole reaction (sensitivity from 1 to 20 μg) to assay the carboxyl groups of the uronic acid for heparin sulfate, chondroitin sulfate and dermatan sulfate and/or anthrone reaction to assay the hexose group for keratan sulfate (Mort and Roughley, 2007).
  7. The DMMB can also be very efficient when performing chromatography to rapidly assay for fractions containing GAGs (Burton-Wurster et al., 2003).
  8. The DMMB-GAG complex that is formed results in the immediate formation of turbidity, however this complex starts to precipitate within 10 min, therefore the absorbance measurement should be performed immediately.

Recipes

  1. DMMB reagent
    Dissolve 16 mg DMMB, 3.04 g glycine, 1.6 g NaCl and 95 ml of 0.1 M acetic acid and complete the volume to 1 L
    Filter (0.45 μm)
    Protect from light
    Do not use if precipitate is present in the solution

Acknowledgments

This protocol was adapted or modified from Farndale et al. (1982) and Whitley et al. (1989).

References

  1. Anwari, K., Webb, C. T., Poggio, S., Perry, A. J., Belousoff, M., Celik, N., Ramm, G., Lovering, A., Sockett, R. E., Smit, J., Jacobs-Wagner, C. and Lithgow, T. (2012). The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex. Mol Microbiol 84(5): 832-844.
  2. Burton-Wurster, N., Liu, W., Matthews, G. L., Lust, G., Roughley, P. J., Glant, T. T. and Cs-Szabo, G. (2003). TGF beta 1 and biglycan, decorin, and fibromodulin metabolism in canine cartilage. Osteoarthritis Cartilage 11(3): 167-176.
  3. Chandrasekhar, S., Esterman, M. A. and Hoffman, H. A. (1987). Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal Biochem 161(1): 103-108.
  4. Farndale, R. W., Sayers, C. A. and Barrett, A. J. (1982). A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 9(4): 247-248.
  5. Mort, J. S. and Roughley, P. J. (2007). Measurement of glycosaminoglycan release from cartilage explants. Methods Mol Med 135: 201-209.
  6. Warren, S. (2000). A critical analysis of the 1, 9-dimethylmethylene blue assay for sulfated glycosaminoglycans in Synovial fluid. University of Guelph.
  7. Whitley, C., Ridnour, M., Draper, K., Dutton, C. and Neglia, J. (1989). Diagnostic test for mucopolysaccharidosis. I. direct method for quantifying excessive urinary glycosaminoglycan excretion. Clin Chem 35(3): 374-379.
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Coulson-Thomas, V. . and Gesteira, T. F. (2014). Dimethylmethylene Blue Assay (DMMB). Bio-protocol 4(18): e1236. DOI: 10.21769/BioProtoc.1236.
Q&A

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DENNIS ROJAS BARILLAS
DENNIS ROJAS BARILLAS
UNMSM
Thank for your protocol first.
I am making separations of mixtures of chondroitin and hiluronic acid (hyaluronate) and I want to apply the protocol to separate these molecules, for which I need to precipitate all the chondroitin present in the mixture and keep the supernatant with hyaluronate. So the question is: How could a total precipitate of chondroitin be achieved? If you know of any methodology that does not involve the use of enzymes for the quantification of hyaluronate in mixtures with chondroitin.

Thank you very much. Greetings.
5/8/2018 12:36:13 PM Reply
Wenhao Ma
Wenhao Ma
the university of technology
Thank for your protocol first.
However, the protocol couldn't help me dealing with the mice urine sample.
First, your protocol mentioned about Tris-base, but I didn't find out where to use it.
Second, I made 10x DMMB solution and dilute with ddH2O to 1x concentration, and I use mice urine sample(20 ul) mixing with 200ul 1x DMMB solution. After reading,there was no changing with data, even the standard curve has not worked. Some mice model urine sample's data should be higher than the control, But not.
I am looking forward your help
Thank you.
4/4/2018 7:54:03 AM Reply
Vivien J. Coulson-Thomas
Vivien J. Coulson-Thomas
University of Houston

First of all you mentioned your standard curve has also not changed color. That would mean that the DMMB solution would not have been prepared correctly.
Second, please read through the notes. Using samples with high salt and albumin content may affect the assay; I am not sure what mouse model you are using to collect your urine samples. Sometimes diluting samples 1:10 can help, especially if you believe the concentration of sulfated GAGs in your samples will fall above the standard curve. You could also add a gel filtration step before the DMMB assay.
Also, in the notes we mention that DMMB requires the length of glycosaminoglycan chain be over a tetrasaccharide. GAGs present in the urine are significantly smaller than those present in tissues, so please make sure your samples are appropriate for this assay.

4/4/2018 10:23:15 AM

Sarita Mishra
Sarita Mishra
IGIB
Hi I have tried the same protocol as mentioned but the standard curve itself looks like the image attached. I have used the same concentrations as mentioned here. Kindly help me if I need to change something. Thanks.
1/11/2018 1:08:22 AM Reply
Matteo Beretta
Matteo Beretta
Fondazione Ca' Granda - Ospedale Maggiore Policlinico
Dear Sir, Dear Madame,

I tried to use your protocol for GAGs quantification in cell colture medium conditioned by intervertebral disc cells for 24h-48h and used the bovine chondroitin 4-sulfate as standard. I am having huge problems even with the standard curve (a eight point-curve in the range 10 ug/ml - 0 ug/ml) since the curve appears linear when prepared with H2O, whereas the machine read almost the same absorbance value at 252nm when the curve is prepared with DMEM+10%FBS or DMEM+0.5%FBS.
Could you give me your opinion about this problem and any possible explanation/solution to that, please?

P.S. I saw that some authors use to digest their samples with papain before the GAGs quantification. Could it help me somehow? And in case yes, do you recommend a particular papain solution and digestion time?

I thank you very much in advance and wish you all a good day.

Kind regards,

MB
6/22/2017 4:53:20 AM Reply
tarsis ferreira
tarsis ferreira
OPTIMVIA

Assuming you media contains L-Cysteine, add EDTA to 0.1 mM and Papain, 0.5 U/mg (Sigma, Cat. No. 76216 - 25 U/ml). Digest samples in papain solution for 15–18 hours at 65°C. Digested samples can be stored at −20°C.

6/22/2017 4:01:18 PM

Matteo Beretta
Matteo Beretta
Fondazione Ca' Granda - Ospedale Maggiore Policlinico

I want to thank you very much for your quick answer!

So do you think that the problem is the L-Cysteine and not the FBS? In light of your scientific experience could you briefly explain me why L-Cysteine could be a problem for the GAGs quantification with the DMMB solution?

Thank you very much again for your help! It's very much appreciated!

Kind regards,

MB

6/23/2017 2:04:34 AM

Mikhail Olinevich
Mikhail Olinevich
Biomed
Dear sir/madame,

I've followed the exact protocol to find sulfated glycosaminoglycans in human skin samples which have been degraded using pronase. A kit from "Biocolour, Blyscan" quantifies the sGAGs in our sample in a proper way. Because of its expensiveness, we have decided to make the dimethylmethyleneblue reagens by ourselfs. The problem that we are experiencing is that no precipitate occurs. By adding some leftovers of the dye reagent from Blyscan, the precipitate immediately follows. How could you explain this problem? Why isn't it working? I suppose it has to do something with the reagent...
The second problem is that there is no variation in absorbance between the different standard concentrations. The absorbance reader (tried at 540 and 510 nm) gives approximately the same value for absorbance for different concentration of chondroitin-4S standard (see photo attachment). I suppose, this problem is also linked to the first one.

At last, in material and reagents, you mention that you need Tris base, but you actually don't use it in the protocol..

Thank you in advance for you help.
Mikhail
2/23/2016 12:34:28 AM Reply
Vivien J. Coulson-Thomas
Vivien J. Coulson-Thomas
University of Houston

Dear Mikhail,

I am not sure if I understood the extent of the problem with the standard curve. Were 3, 4 and 5 ng shown in your photo done with the reagent prepared based on our protocol? They seem to have worked. They are very close points on the curve so maybe you could open the curve a little. Use 1.25 microg/mL, 5 microg/mL and 10 microg/mL while testing.
Now about your samples, what are they and what were they prepared in? Some detergents or high salts like NaCl and/or Urea can affect the DMB reaction. Could you please provide more information on how these samples were purified?


Thank you.



3/2/2016 9:10:38 AM

Mahadevan R
Mahadevan R
Dept of Biochemistry
Sir, can I use this method to detect the sulfated GAG in a decellularized ECM? For that do I have to extract the sulfated GAG first or can I dissolve the decellularized ECM in

Sir, I have extracted collagen from fish skin. It has a
10/14/2015 2:17:29 AM Reply
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