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Background correction when reading ELISA with TMB substrate
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Neela Fazl
Background correction when reading ELISA with TMB substrate
Using different wavelength for reference give some advantages. You can measure a baseline of each well. All well might not have the same baseline because each well might not be the exactly same as others. In addition, you could make scratches on it, you might touch the bottom, or you get some dirt from your bench. And this can be corrected by A540 in your case. So, variations at a lower range are not much concerned. Sometimes, even if you choose a wavelength where the absorbance seems not to change after reaction, there might be a small change. This is not concerned ether, because both absorbances are concentration dependent unless states in solution do not change (froming complexes or something like that).
But still, you get high background from samples showing 1.4 or more at A450. I do not have experiences for this substrate, but you might want to think several possibility:
1)You might get bubbles on the top of your solution in the wells,
2)You might not get accurate values because OD is too high,
3)The solution might get precipitates
4)This may be a very less possibility, but some secondary reaction occurs.
For 2), 3), and 4). You might want to check linearity by drawing a standard curve, and avoid to use the range deviating from the liner relation.
PS
I should've mention that you would check the specification of your plate reader. OD=3 could be way to high to measure it. Because light intensity decreases to one thousandth though 1 cm light path at OD=3. It is very hard to measure such weak lights exactly. Probably many specifications of plate readers say you could measure ODs up to around 2, but some people think it is ideal to measure ODs up to around 1.
Using different wavelength for reference give some advantages. You can measure a baseline of each well. All well might not have the same baseline because each well might not be the exactly same as others. In addition, you could make scratches on it, you might touch the bottom, or you get some dirt from your bench. And this can be corrected by A540 in your case. So, variations at a lower range are not much concerned. Sometimes, even if you choose a wavelength where the absorbance seems not to change after reaction, there might be a small change. This is not concerned ether, because both absorbances are concentration dependent unless states in solution do not change (froming complexes or something like that).
But still, you get high background from samples showing 1.4 or more at A450. I do not have experiences for this substrate, but you might want to think several possibility:1)You might get bubbles on the top of your solution in the wells,2)You might not get accurate values because OD is too high,3)The solution might get precipitates4)This may be a very less possibility, but some secondary reaction occurs.
For 2), 3), and 4). You might want to check linearity by drawing a standard curve, and avoid to use the range deviating from the liner relation.
PSI should've mention that you would check the specification of your plate reader. OD=3 could be way to high to measure it. Because light intensity decreases to one thousandth though 1 cm light path at OD=3. It is very hard to measure such weak lights exactly. Probably many specifications of plate readers say you could measure ODs up to around 2, but some people think it is ideal to measure ODs up to around 1.
Using different wavelength for reference give some advantages. You can measure a baseline of each well. All well might not have the same baseline because each well might not be the exactly same as others. In addition, you could make scratches on it, you might touch the bottom, or you get some dirt from your bench. And this can be corrected by A540 in your case. So, variations at a lower range are not much concerned. Sometimes, even if you choose a wavelength where the absorbance seems not to change after reaction, there might be a small change. This is not concerned ether, because both absorbances are concentration dependent unless states in solution do not change (froming complexes or something like that).
But still, you get high background from samples showing 1.4 or more at A450. I do not have experiences for this substrate, but you might want to think several possibility: 1)You might get bubbles on the top of your solution in the wells, 2)You might not get accurate values because OD is too high, 3)The solution might get precipitates 4)This may be a very less possibility, but some secondary reaction occurs.
For 2), 3), and 4). You might want to check linearity by drawing a standard curve, and avoid to use the range deviating from the liner relation.
PS I should've mention that you would check the specification of your plate reader. OD=3 could be way to high to measure it. Because light intensity decreases to one thousandth though 1 cm light path at OD=3. It is very hard to measure such weak lights exactly. Probably many specifications of plate readers say you could measure ODs up to around 2, but some people think it is ideal to measure ODs up to around 1.
Using different wavelength for reference give some advantages. You can measure a baseline of each well. All well might not have the same baseline because each well might not be the exactly same as others. In addition, you could make scratches on it, you might touch the bottom, or you get some dirt from your bench. And this can be corrected by A540 in your case. So, variations at a lower range are not much concerned. Sometimes, even if you choose a wavelength where the absorbance seems not to change after reaction, there might be a small change. This is not concerned ether, because both absorbances are concentration dependent unless states in solution do not change (froming complexes or something like that).
But still, you get high background from samples showing 1.4 or more at A450. I do not have experiences for this substrate, but you might want to think several possibility:1)You might get bubbles on the top of your solution in the wells,2)You might not get accurate values because OD is too high,3)The solution might get precipitates4)This may be a very less possibility, but some secondary reaction occurs.
For 2), 3), and 4). You might want to check linearity by drawing a standard curve, and avoid to use the range deviating from the liner relation.
PSI should've mention that you would check the specification of your plate reader. OD=3 could be way to high to measure it. Because light intensity decreases to one thousandth though 1 cm light path at OD=3. It is very hard to measure such weak lights exactly. Probably many specifications of plate readers say you could measure ODs up to around 2, but some people think it is ideal to measure ODs up to around 1.
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