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Elisa troubleshooting

Elisa troubleshooting - Biology Forum

Elisa troubleshooting - Biology Forums. Ask questions and discuss the study of Biology. If you have Biology questions from your homework ask them here!


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  #1  
Old 06-28-2013, 06:05 AM
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Default Elisa troubleshooting



Hello!
Nice to be on this forum!
I would like to know something that I can't find on the internet no matter what I do:
1. LEt's suppose that you're doing an Elisa test, and positives lose color more and more, after adding the stop solution, until, after 3-4 minutes they are completely blank (transparent).Why does it happen?
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Old 06-28-2013, 06:07 AM
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Default Re: Elisa troubleshooting

It's indirect Elisa, btw.
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Old 06-28-2013, 12:21 PM
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Default Re: Elisa troubleshooting

Which substrate and stopping solution you use?
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Old 07-01-2013, 04:45 AM
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Default Re: Elisa troubleshooting

TMB as substrate and 1N HCl as stopping solution.
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Old 07-02-2013, 05:54 AM
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Default Re: Elisa troubleshooting

Stopping solutions of the TMB substrate
TMB has been shown to be a safe-sensitive substrate for the assay of horseradish peroxidase (HRP). Initially, in the presence of HRP and hydrogen peroxide, a one-electron oxidation product is formed. This compound, a cation free radical, is blue in colour with an adsorption maximum at 653 nm. Further reaction with HRP/H2O2 or acidification of the radical with acid yields the diimine terminal oxidation product yellow with an absorbance peak at 450 nm. The extinction coefficient of the radical (Abs653 nm = 3.9 x 10^4 mol^-1 cm^-1) and diimine (Abs450 nm = 5.9 x 10^4 mol^-1 cm^-1) provide a remarkably sensitive system for the assay of HRP and HRP labelled probes. Several acid solutions may be used to stop the TMB reaction. A recommended acid stop solution is 1 N H2SO4 or a mixture of HCl and H2SO4 (final concentration equals 1 N HCl and 3 N H2SO4) or with sodium fluoride. In endpoint assays, the substrate reaction can be stopped with 1 N HCl or 0.6 N sulfuric acid. The intensity of the acidified reaction product may change over time. For best results, specially when used 1 N HCl, to read immediately after adding acid stop solution. We have observed that 0.5 mol/L-1 hydrochloric acid (adding 83 ml of 6 mol/L AR HCl to 917 ml of reagent grade water must contain less than 10-7 mol/L^-1 of iron or copper salts otherwise unreacted TMB will be converted non-enzymatically to the diimine) converts the blue radical to the yellow diimine and the yellow colour is stable for at least 30 minutes after the addition of acid. Same result has been obtained employing a 0.3 - 1 M phosphoric acid (H3PO4) solution provides the most stable endpoint color.
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Old 07-22-2013, 07:29 AM
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Default Re: Elisa troubleshooting

Well, I am studying Biotechnology. As I found the one of the best firm on life science research i.e bosterbio.com. You can also recommend them. I am sure, here you got your answer.
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