I am using Dot Blot study to detect 5hmC level. Dot blot detection is ok but when I use methylene blue stain to check the control, the dots are not so clear. Very faint color I can see on the membrane. Two methylene blue staining methods I used but result is same.
Method-1: Membrane is washed with miliQ water and then treated with 0.04% methylene blue in 0.5M sodium acetate (pH 5.2) for over night.
Method-2: Membrane is washed with miliQ water and then treated with 5% acetic acid solution for 15 min at room temperature. After that treatment with 0.04% methylene blue in 0.5M sodium acetate (pH 5.2) for over night.
Is there anybody who can give some valuable suggestions that could help me to solve this problem?
I don't know this specific procedure, but it can be a good start to check, if the dye is the right one. Sometimes Methyl-blue, Methylene-blue and anilinblue are confused. They have different staining characteristics. One should look for the right CI-number of the reagens.
http://stainsfile.info/StainsFile/dyes/dyes.htm
Staining procedures with acid solutions of the dye use mostly methyl-blue CI 42780 (anilinblue as synonym). Methyleneblue CI 52015 is found in Loefflers methylenblue as counterstain in Ziehl-Neelson-staining. Loefflers methylenblue is an alkaline solution. CI 52015 is also ingredient of Giemsa for DNA-staining.
To increase staining it may be helpful, just to increase the dye-concentration. An overstaining can be removed by longer washes.
Stain about 5 min with 0.1 % new methylene blue N (Sigma) in 23% ethanol, 0.2 mM sodium hydroxide, followed by destaining with water. Change water several times.
Thank you very much Dr. Donal J Chabot. I will try this protocol.
@ Dr. Donal J Chabot,
I have tried your suggested protocol but I was failed. Is there any specific factors to get success in this protocol?
what about this?
http://mrcgene.com/wp-content/uploads/2014/06/MethyleneBlue-June-2014.pdf
We actually do 5hmC staining and methylene blue separately. We used non denatured DNA for internal control methylene blue staining. It worked quite well. You can try if this works for you.
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