I am trying to measure arginase activity in serum samples from cancer patients and in cancer cell lysates. I am using the colorimetric assay according to Corraliza et al (in brief: I take 22,5 ul serum , add 1,25 ul Tris-HCl (endconc. 50 mM) and 1,25 ul MnCl2 (endconc. 10mM), heat for 10 min 55C, then add 25 ul 0.5M L-arginine, incubate 1hr 37C, stop reaction adding 200 ul acid solution mixture (H2SO:H3PO4:H2O 1:3:7), then add 25 ul 9% alpha-isonitrosopropiophenone (in 100% EtOH), heat for 30 min 95C, and read after 10 min at 540 nm. My problem is, that after the 1hr incubation at 37C I get in all samples (even in the blank) a brown precipitate- I guess it is some Mn-complex. But this is not the biggest problem (this precipitate gets diluted after the acid addition and dissapear later, worse is, that after the 95C heating, when I get the violet color, I get in the serum samples (much less in the cell lysates) a heavy, cloudy, white precipitate (reminds me of denatured proteins when you lyse cells for DNA-isolation), which I even cannot centrifuge down. It is almost impossible to pipet the sample into the 96-well plate for reading without transfering this precipitate - and this gives a great mess during the reading. I also tried to use a commercial urea kit (from Abnova) to measure the urea after the 1hr incubation at 37C (just added the working solution from the kit). But here I have the problem, that after adding L-arginine I get lower OD than when I add just water (the same situation when I add L-arg to the blank) - as if L-arg somehow blocks the colorimetric reaction. Just measuring the urea (without addition of L-arg) is working fine. Does anyone have similar expierience and have any advice?
Hello Malgorzata,
I'm a fellow in Tom Wynn's lab at the NIH, and we routinely test arginase activity in cell and tissue lysates using a different technique than what you describe. You could try adapting this approach to your experiment as an alternative method.
http://www.ncbi.nlm.nih.gov/pubmed/21462164
I've also noticed a brown, manganese-containing percipitate (which I find doesn't change the results), but I've never seen anything like the cloudy white percipitate you describe. Perhaps the pH of your samples is involved? The arginase enzymatic reaction is faster at a high pH, and I presume your L-arginine solution has a pH of 9-10. As a guess, try adding water instead of arginine to your sample and see whether the cloudy white percipitate still forms. If not, you may be able to run your assay on serum using less substrate.
Good luck,
Luke
Manganese forms Brown precipitate at higher pH. It is advisable to first dislove manganese in little amount of deionized water and and then make up the volume with buffer.
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