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

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i had used Bradford method(Coomassie brilliant blue G-250) to assay proteins in urine,it seems good.
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no not specific proteins. i am just looking for proteinuria...

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

Are you looking for particular proteins? Gammopathies?


hi :lol:

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

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

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


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.

-Zagami Francesco-
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