amount of proteins in urine - (Jul/05/2009 )

i had used Bradford method(Coomassie brilliant blue G-250) to assay proteins in urine，it seems good.

no not specific proteins. i am just looking for proteinuria...

sgt4boston on Aug 17 2009, 11:27 AM said:

hi

did u dilute the urine sample if yes then how much dilution?

chdd on Aug 20 2009, 12:26 PM said:

I measure microalbuminuria by means a screening spot test (SST) on cellulose acetate employing 2 μl of random fresh emitted (<2 h) or stored at 4 °C urine specimens that are spotted on dry cellulose acetate and allow to stay for 2-3 min until spot area is completely dry. Immerse cellulose acetate film in dye CBB R-250 solution (dissolve in glass amber bottle, 100 mg of Coomassie R-250 in 22.5 ml of methanol, mixing from time to time for inversion for then minutes, and add 5.0 ml acetic acid, mix as above, and finally add 22.5 ml of dH2O, mix as above and allows to rest for at least 5 hrs before use. Stable 1 year at room temperature) and incubate for 4 min. at RT. Draw and drain the film and to set it in destain solution (5% acetic acid aqueous solution) for 3 cycles of 10 min. Spot positive is underlined by different intensity blue color proportional to albuminuria concentration, while the uncolored spot area identified the specimen as normal value. Calibration is carried out using human serum albumin saline solutions ranging from 20 to 200 mg/L. Starting from albumin standard 60 g/L (60000 mg/L) dilute 1:100 (100 μl + 9.9 ml) with normal saline solution (NSS - 0.9% NaCl) obtaining standard 600 mg/L and from this made decreasing serial dilution always in normal saline as below indicated :
300 mg/L : 500 μl 600 mg/L ST + 500 μl NSS; 200 mg/L : 400 μl 300 mg/L + 200 μl NSS; 150 mg/L : 300 μl 200 mg/L ST + 100 μl NSS; 100 mg/L : 300 μl 150 mg/L + 150 μl NSS; 75 mg/L : 300 μl 100 mg/L + 100 μl NSS; 50 mg/L : 200 μl 75 mg/L ST + 100 μl NSS; 30 mg/L : 150 μl 50 mg/L + 100 μl NSS; 20 mg/L : 100 μl 30 mg/L + 50 μl NSS. For test screening compare the color intensity spot of the sample with that of the serial calibration range.

Proteinuria screening : dissolve 0.1 g of 3’,3”,5’,5”-tetrabromophenolphthalein ethyl ester potassium salt (Sigma cod. T1889) in 100 ml tert-butanol (Fluka cod. 19470), and 8 ml glacial acetic acid (Merck cod. 1000063) is added, resulting stable 1 year when stored, strictly capped, at room temperature.  In ELISA microplate (BioHit), two drops or 80 µl of this solution added to urine (5 drops or 200 µl) containing the same amount of albumin as the albumin containing urines (5, 10, 20, 30, 50, 75 and 100 mg/dl, in which values between 10-20 mg/dl are cut off for pathological results) produced a blue color, with color intensity proportional at albumin concentration, that can be read at microplate reader spectrophotometer at 630 nm.

Urine proteins determination : transfer in centrifuge tube, 0.5 ml of filtered or centrifuged urine, add 0.5 ml of 1.5 M NaCl, and 0.5 ml of 1 mM tannic acid in dH2O, containing 1 g/L of benzoic acid, stable for two months at RT. Mix, and after 5 min centrifuge for 10 mm at 3000 rpm. Decant the supernatant liquid and invert the tube on filter paper to drain thoroughly. Add 5.0 ml of 1.5 M NaCl and, after suspending the precipitate, centrifuge and remove the wash solution as above. Add 2.0 ml of dH2O and 0.5 ml of the ferric chloride reagent (dissolve 10 mM ferric chloride in a mixture formed by equal volumes of triethanolamine and dH2O. Stable for two months at RT) to the precipitate, and mix. After 5 min., Abs is measured at 510 nm, within 4 hours, against a blank consisting of 2 ml of dH2O and 0.5 ml of ferric chloride reagent, calculating result versus series standards of albumin (0.05 to 2.0 g/L) obtained diluting in 0.85% NaCl, stock standard  100 g/L in 0.85% NaCl, with linearity up to 1.5 g/L. For samples with higher concentration repeat the determination with urine diluted 10 fold in physiological saline. The quantity of protein so recovered from urine of normal subjects was found to range from 70 to 150 mg/L