Lidstrom:Solution Stock Info: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: Back home: Lidstrom:Protocols NADH * NAD(P)H are rapidly destroyed in acid in conditions where NAD(P)+ are completely intact *99% destruction of NADH **@ 23 deg C =) |
No edit summary |
||
| Line 2: | Line 2: | ||
==NAD(P)/H== | |||
* NAD(P)H are rapidly destroyed in acid in conditions where NAD(P)+ are completely intact | * NAD(P)H are rapidly destroyed in acid in conditions where NAD(P)+ are completely intact | ||
*99% destruction of NADH | *99% destruction of NADH | ||
**@ | **@23°C = 1.2 min @ pH 2, = 2 hr @ pH 4 | ||
**@38°C, 3-4x faster | |||
**@60°C, 20x faster | |||
*NADPH is destroyed faster than NADH (~80% faster @ 30°C) | |||
*@pH 2, NAD+ is 100,00x more stable than NADH | |||
** Allows elimination of reduced form by pH drop | |||
* Above pH 7, degradation rate of NAD increases,5000x faster at pH 12.5-13 | |||
* NADP+ appears to be more stable than NAD+ | |||
* Higher temp, more salts increase degradation rate | |||
*NADH becomes more stable with pH increases, excluding low concentration in small volumes | |||
*Oxidation is a problem above pH 8 | |||
*@ pH 7 25°C, rate of destruction in ~0.2%/hr, increases 10x @ pH 6 | |||
*Strong solutions (40 mM) of NADH store well @ pH 9-12, but large losses @ 4°C | |||
*Weak solutions (0.4 mM) of NADH store well @ 4°C from pH 9-11, destroyed @ -20°C pH >10.5 | |||
*Similar characteristics for NADPH | |||
*NADH presence in small volumes is sensitive to oxidation at neutral pH | |||
** increases ~inverse square root of the volume | |||
** can prevent with 1 to 2 mM ascorbic acid | |||
=Recommendations= | |||
*Store pyridine nucleotides, desiccated @ -20°C | |||
*Can store NAD+/NADP+ solution for months without significant concentration change/ | |||
*Can standardize stock solutions with spectrophotometer | |||
*Dissolve NAD+/NADP+ in water, ~100 mM | |||
*Unless < -40°C, NAD(P)H should be prepared at < 5 mM, pH 9-11 and stored at 4°C. At low temp, strong solution can probably be stored without too much loss | |||
Enzymatic Analysis: A pract | |||
Revision as of 16:25, 5 December 2013
Back home: Lidstrom:Protocols
NAD(P)/H
- NAD(P)H are rapidly destroyed in acid in conditions where NAD(P)+ are completely intact
- 99% destruction of NADH
- @23°C = 1.2 min @ pH 2, = 2 hr @ pH 4
- @38°C, 3-4x faster
- @60°C, 20x faster
- NADPH is destroyed faster than NADH (~80% faster @ 30°C)
- @pH 2, NAD+ is 100,00x more stable than NADH
- Allows elimination of reduced form by pH drop
- Above pH 7, degradation rate of NAD increases,5000x faster at pH 12.5-13
- NADP+ appears to be more stable than NAD+
- Higher temp, more salts increase degradation rate
- NADH becomes more stable with pH increases, excluding low concentration in small volumes
- Oxidation is a problem above pH 8
- @ pH 7 25°C, rate of destruction in ~0.2%/hr, increases 10x @ pH 6
- Strong solutions (40 mM) of NADH store well @ pH 9-12, but large losses @ 4°C
- Weak solutions (0.4 mM) of NADH store well @ 4°C from pH 9-11, destroyed @ -20°C pH >10.5
- Similar characteristics for NADPH
- NADH presence in small volumes is sensitive to oxidation at neutral pH
- increases ~inverse square root of the volume
- can prevent with 1 to 2 mM ascorbic acid
Recommendations
- Store pyridine nucleotides, desiccated @ -20°C
- Can store NAD+/NADP+ solution for months without significant concentration change/
- Can standardize stock solutions with spectrophotometer
- Dissolve NAD+/NADP+ in water, ~100 mM
- Unless < -40°C, NAD(P)H should be prepared at < 5 mM, pH 9-11 and stored at 4°C. At low temp, strong solution can probably be stored without too much loss
Enzymatic Analysis: A pract
