Dopamine Measurment in PC12
I am measuring Dopamine release from NGF-differentiated PC12. I am using
HPLC with C18 reverse phase column in 10 % methanol isocratic elution.
It seem that after 3-4 times the column get clotted, the pressure go high
and we need to wash a lot with metanol (much more than regular
wash). Sometimes the injection needle is blocked and it takes time to
Is anyone knows a better way to wash the column after meausiring dopamine
from PC12 supernatant?
In my lab. some people measure norepinephrine from synaptosomes and after
measurment HPLC works well without problems.
Dopamine Measurment in PC12
seems to me you get a precipitate that blocks the system.
a) filter your sample through 0.2µM syringe filters
b) bring your sample to 10% MeOH and spin any precipitating material
down before injecting (better before step a) if you consider b)
c) use SPE columns for sample cleanup. Should work with methods for
catecholamine isolation from blood. You might refer to the clinical
chemistry department of your favorite hospital
d) use a small column of the same type as pre-column to protect the
actual HPLC column
e) reverse your column every 2nd or so run.
Primer extension kinetics
Does anybody know the kinetics of primer extension reaction? Is it
A related question is, under the temperature of a primer extension
reaction, does the primer-template hybrid gets disassociated? If they
do, to what extent, in other words, what magnitude is K as compared to
k in the primer extension step.
Thanks a lot.
Re: Dopamine Measurment in PC12
Colorimetric dopamine measurement
The method, applied to the determination of dopamine (DA) in human serum samples, is based on the reaction of dopamine with the oxidizing agent (silver nitrate) in the presence of PVP (as a stabilizer) and the formation of silver nanoparticles in a slightly basic medium. Spectrophotometry is used to monitor the changes of the surface plasmon resonance (SPR) band at a maximum wavelength of silver nanoparticles (440 nm) vs. time.
Silver nitrate stock solutions (SNS) 0.01 M : dissolve 0.085 g AgNO3 in 5 ml dH2O and after diluting to 50 ml.
PVP stock solution : dissolve 0.4 g/L polyvinylpyrrolidone by average mol wt 10000 in 5 ml dH2O and after diluting to 25 ml.
Standard dopamine 0.05 M :dissolve daily mg di 3-hydroxytyraminium chloride (Dopamine) in dH2O
Alkaline solution : dissolve 0.001 M NaOH to adjusted of pH reagent solution
Dispense in 5 ml tube, 1 ml SNS, 0.7 ml of PVP, 2.3 ml of different concentrations of the dopamine and 1 ml of alkaline solution to obtain the reasonable solution. Then it was mixed slowly and a portion of that was transferred within 7 min into a 1 cm spectrophotometric cell to record the absorbance. It should be noted that the order of the addition of the reagents is very important. The absorbances were measured at 440 nm that is λ max of silver nanoparticles surface plasmon resonance peak at this condition, against a reagent blank. Linear calibration graph is obtained in the concentration range of 3.2×10^–6 - 2.0×10^–5 mol/L–1, and with detection limits of 8.0×10^−7 which are comparable by different spectrophotometric and colorimetric methods.
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