ATPase staining for different Myosins - Anyone got a good protocol? (Feb/18/2009 )
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I am going to stain cryosectioned slides of skeletal muscle for different myosin heavy chains. I have found a few protocols on the ATPase method for pH 9.8 , 4.3 , 4.5 and 4.6 to stain the different forms (2A, 2B and 2X and type 1). In some of the protocols people have used Sodium barbital, I have checcked around and it appears to be a narcotic substance under heavy regulations. Is it possible to use anything else for this staining? Is there any replacement for this substance? What does it do and why would you need it in a simple staining protocol?
mdfenko on Feb 18 2009, 06:19 PM said:
I checked it through but frankly I cannot make full sense of it since it only deals with the High pH staining, I wonder if it is the same for the lower pH stainings... I am looking for a protocol that does not require barbital at all...
barbital (veronal; diethyl barbituric acid, sodium salt) has a pK of 7.8 (or 7.98, depending where you look). it would not have been used for low pH work. why would you be looking for a substitute for something you wouldn't have been using in the first place?
was the buffer being used made with barbituric acid? barbituric acid has a pK of 4 and would be suitable for low pH work (~3-5).
you can try acetate buffer (i wouldn't use phosphate buffer with atpase).