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-   -   Dopamine Measurment in PC12 (http://www.molecularstation.com/forum/protocols-methods-forum/65091-dopamine-measurment-pc12.html)

Federico Corti 03-10-2009 04:09 PM

Dopamine Measurment in PC12
 
Dear members,

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
clean it.
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.

Thanks

Fede

WS 03-10-2009 06:08 PM

Dopamine Measurment in PC12
 
Hi Federico,

seems to me you get a precipitate that blocks the system.

you could

a) filter your sample through 0.2M 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.

Best regards,

Wo

Wen Huang 05-08-2009 08:09 PM

Primer extension kinetics
 
Hi

Does anybody know the kinetics of primer extension reaction? Is it
linear?

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.

Wen


Zagami 05-26-2013 06:20 PM

Re: Dopamine Measurment in PC12
 
Colorimetric dopamine measurement
Principle
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.
Reagents
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
Procedure
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 3.210^–6 - 2.010^–5 mol/L–1, and with detection limits of 8.010^−7 which are comparable by different spectrophotometric and colorimetric methods.


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