Cells plated in 12 wells plates were grown to confluence. When cells were preincubated with 1 mM unlabeled NAD+ for 24 h, the wells were washed three times with 1 ml of PBS and then incubated in a buffer solution (140 mM NaCl, 10 mM Hepes/Na, 2.5 mM MgSO4, 2 mM CaCl2, and 5 mM KCl, 5 mM glucose, pH 7.4) containing 500 pM [14C]NAD+ (54 mCi/mmol), [3H]NAD+ (28.6 Ci/mmol), or [14C]AMP (60 mCi/mmol) (PerkinElmer). Competing compounds and drugs were added to the wells 15 min before labeled molecules unless stated in the text. After 10 min, cells were washed three times with 1 ml of PBS and lysed with 0.5 M NaOH. Incorporated radioactivity was assayed by liquid scintillation counting.
3h nad
Manufactured by PerkinElmer
[3H]NAD+ is a radiolabeled nicotinamide adenine dinucleotide (NAD+) compound, where the hydrogen atom at the C-4 position of the nicotinamide ring is replaced with a tritium (3H) isotope. NAD+ is an essential cofactor involved in numerous redox reactions in living organisms.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using 3h nad
1
NAD+ Uptake and Metabolism in Cells
2
Measurement of cADPR and NAADP levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with 0.5 mL of 0.6 M perchloric acid under sonication, and precipitates were removed by centrifugation at 20, 000 x g for 10 min. Perchloric acid was removed by mixing the aqueous sample with a solution containing three volumes of 2 M KHCO 3 . After centrifugation at 15, 000 x g for 10 min, the aqueous layer was collected and neutralized with 20 mM sodium phosphate (pH 8) [ cADPR] i and [NAADP] i were measured using a cyclic enzymatic assay as described previously [28, 29] .
[ 3 H ] cADPR binding assay [ 3 H] cADPR was synthesized by Aplasia cyclase using [ 3 H] NAD (PerkinElmer) and purified by HPLC [30] . To examine the binding of [ 3 H] cADPR to SERCA, GST-labeled recombinant 50 ng/ml SERCA (Abnova) were attached to GST magnetic beads. 10 pM [ 3 H] cADPR binding to recombinant proteins were quantified in the absence and presence of 10 µM cADPR.
[ 3 H ] cADPR binding assay [ 3 H] cADPR was synthesized by Aplasia cyclase using [ 3 H] NAD (PerkinElmer) and purified by HPLC [30] . To examine the binding of [ 3 H] cADPR to SERCA, GST-labeled recombinant 50 ng/ml SERCA (Abnova) were attached to GST magnetic beads. 10 pM [ 3 H] cADPR binding to recombinant proteins were quantified in the absence and presence of 10 µM cADPR.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!