***PLEASE HELP with NADH***

X-Man bike at wpi.edu
Mon Aug 1 20:03:36 EST 1994


We are looking for assistance concerning an assay and NADH.  I am attempting
to duplicate an assay as performed by LANE et al. 1969 concerning PEPCK in
Limulus.  I raise concern with the use of the  NADH.  We are using vials of
premeasured NADH (2.56umoles/vial).  My final solution contains the following
amounts:
 
 	KHCO3		50 ul
 	PEP	       100 ul
 	GDP	       100 ul
 	MnCl2		50 ul
 	Mercap(or DTT)	50 ul
 	MDH	       100 ul
      ***NADH		50 ul ***
       ________________________
 		       500 ul

Since the NADH is 1/10 of the solution I don't know if the concentration will
change or even which way is better to combine it.  I could either put it
into solution by combining it with H2O and then adding that to the solution
or by adding the liquid solution to the vials.

If anyone could help on which is preferable, and how to calculate if we
add the solution to the vials it would be greatly appreciated.  

Thank you for any assistance in advance!!!!!
  
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