Hi, I am stuggling with the method described in the paper published by Reddy KG -1996. I dont see any difference between my highest stock concentration standard and my blank which is nothing but water. I dont understand what is going wrong tried making freshstock and fresh chloramine T solution still nothing changed. Any one got any experience with this assay? any suggestions please!!
Thanks
Smitha
What are you using as your standard? You can try hydroxyproline first to make sure the oxidation/DMAB reaction if working, then try collagen to make sure your hydrolysis conditions are right. Its not a difficult assay so it should be easy to identify where the problem is.
thats a simple method .. may be there's a small mistake such as pH of the buffer or something else.
Thans Nick and Shalini for your response. I use 25ul of hydroxyproline as my standard (20-2ug) from 1mg/ml stock which is dissolved in distilled water and stored at 4 deg. I added 25ul of 4N NaOH (to get 2N final conc) and incubate for approx 25 min at 121 deg (alternatively I tried autoclaving at 120 deg but by the time I take the samples out it is almost 55 min). However both the samples got no difference in final reading. The chloramine T solution (freshly made or stored at 4 deg) was added and incubated at room temp for 25 min according to the protocol. Ehrlich reagent made (12mls) fresh every time with 1.5g p-dimethylaminobenzaldehyde dissolved in 8mls n-proponal and 4mls perchloric acid and kept in boling water bath at 65 deg for 20 min. I dont see any colour change till this step. Once I add the Ehrlich reagent I see the solution turns yellow and that stays the same till reading. I read the absorbance at 550nm after transfering 200ul to a plate reader.
The Acetate citrate buffer is stored at room temp and was made alomost a month before. Will that be a problem? Also should I mix hydroxyproline with HCL? Thanks in advance. Sorry cant figure out where I am going wrong. Please help.
I don't use Reddy's protocol but the oxidation reaction to form pyrrole and the subsequent reaction with DMAB should be essentially the same. I dissolve the Chloramine-T in acetate/citrate/isopropanol. The DMAB is dissolved in perchloric acid/isopropanol. Hydrolyzed samples are evaporated to dryness ( you can use your hydroxyproline std at this point). I use 0-0.2-0.4-0.6-0.8-1.0 ug of HP for my standard curve. I add 100 ul for the Chloramine-T reagent, incubate at RT for 5 minutes then 100 ul of the DMAB reagent and incubate at 60°C for 90 minutes to develop the color. Less than 1 hour and the color development is not complete, beyond 2 hours, the background increases too much. This gives me a std curve of roughly 0-1.0 OD at 550 in a 96 well plate.
Looking at your protocol, maybe you need to cook with the DMAB longer to get the color reaction to go. At least in my case it needed much longer than 20 minutes.
have not exactly used Reddy's but of Bergman n loxely, 1963. certain modifications bt more or less same. u can try: 1. keep your acetate/citrate buffer in refrigerator 2. u haven't mentioned pH of the same (I used AC buffer at 6.0 pH) 3. u try incubating hydroxyproline with oxidant solution (i make by dissolving chloramine-T in buffer) at RT (20-25 minutes) followed by incubating with Ehrich's reagent at 60-65 degrees for 20 minutes. let it stand for an hr or so at RT and then take the reading.
May be this would be of some help to find out way to ur problem.
thanks Shalini, I used pH6.5 for my acetate-citrate buffer as mentioned in the paper, and yes i read straight after the 20 min incubation of Ehrich reagent. Will try your method.
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