I'm currently preparing for some in situ hybridisation, which requires Maleic Acid Buffer with Tween (MABT) for some of the wash steps. The recipe i have been given is for 1L of 5x concentrated MAB, but it seems to be taking ages to dissolve. I have to have the solution prepared for autoclaving by 10am as my lab can only autoclave once per day at the moment. Is it normal for MAB to dissolve slowly? Does anyone have any recommendations?
What is the pH that you want to buffer? I would prepare 1 L of 1X buffer if it takes ages to dissolve. If the problem persists I would use the sodium salt of maleic acid and maleic acid to prepare the buffer or add NaOH to help dissolving the maleic acid.
Hi James, personally, I don't know why you are using maleic acid as a buffer. It has a pKa of ~2 and is difficult to work with since it can also act as a chelator and can be oxidize.
There are commercially available buffers that are easier to use for in situ hybridization:
http://cshprotocols.cshlp.org/content/2007/8/pdb.rec11085.full?text_only=true
http://www.enzolifesciences.com/ENZ-33808/in-situ-hybridization-buffer-1.25x-concentrate/
http://www.abcam.com/protocols/ish-in-situ-hybridization-protocol
The protocol you recommend from abcam at the bottom uses Maleic acid buffer (MABT) which is what im trying to make...from what I have seen it is a common buffer used in a lot of protocols for washes post-hybridisation.
I just want to know if from everyone else's experience it is difficult to dissolve everything and takes a while to prepare. I'm fairly confident it should work as the lab i am in has made 10x concentrated Maleic acid buffer. I need to make several litres at once, so i would rather not have to make it at 1x concentration...
The thing is that solubility of malic acid will increase with pH. Thus the final pH is critical for disaolution.
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