Minimum volume needed for measurement of absorbance values in 96 well plate - by using Elisa plate reader (Oct/07/2008 )
Hi,
I would like to know what is the minimum volume that need to be loaded in 96 well plate to obtain the acceptable absorbance value at certain wavelength when we read the plate using Elisa plate reader.
Is it 100 ul? Or can be less than that?
it depends on the shape of the well.
we used 50ul in round well (didn't check if this was minimum).
you can try putting 50ul, 48ul, 46ul....10ul in the wells and then read the plate with the plate reader.
Normally, people use 50ul per well.
we used 50ul in round well (didn't check if this was minimum).
I am using 96 flat well plate. And the color that I need to read was quite light (light yellowish color). I don't know whether small volume will affect the readings..Or as long as it can fill up the surface of the well?
we used 50ul in round well (didn't check if this was minimum).
I am using 96 flat well plate. And the color that I need to read was quite light (light yellowish color). I don't know whether small volume will affect the readings..Or as long as it can fill up the surface of the well?
I don't think it would make any difference to the color of the solution if you fill it up the surface of the well.
If you fill the solution up the surface, you may have a risk of cross contamination between the wells.
Do you have postive control?
Just wondering...
What is the absorbance value for your ELISA plate?
we used 50ul in round well (didn't check if this was minimum).
I am using 96 flat well plate. And the color that I need to read was quite light (light yellowish color). I don't know whether small volume will affect the readings..Or as long as it can fill up the surface of the well?
I don't think it would make any difference to the color of the solution if you fill it up the surface of the well.
If you fill the solution up the surface, you may have a risk of cross contamination between the wells.
Do you have postive control?
Just wondering...
What is the absorbance value for your ELISA plate?
I am going to test with positive control next time, before that without positive control. It's melanin assay. i am measuring the color produced by the melanin from the melanoma cells. The absorbance values increase with increasing cell number. The highest value I managed to get with very confluent well is only about 0.5, I read absorbance value at 400 nm. Is the absorbance value too low? I thik the cells not giving very dark color pigments. Then the positive control is to inhibit the melanin formation, the absorbance value should be lower than the negative control well.
What should I do?
I think the absorbance is too low.
What type of ELISA are you doing? indirect or sandwich?
What type of ELISA are you doing? indirect or sandwich?
It's not ELISA assay, just a colorimetric assay (read the color formed from the melanin pigments), I used the microplate reader to measure the absorbance value ( I think the priciple is same as a spectrophotometer which using cuvette for larger volume).
If its too low, then what is the minimum absorbance value I should get for any colorimetric assay?
You can't tell what the minimum absorbance value is that you need for your test, make a calibrationline with your melanin pigments.
For the best results a OD between 0.1 en 0.5 is favorable. The problem with lower OD is that CV of the measurements will increase.
If the volume is to low you can dilute it with the solvent ,the OD will not change because the lowering concentration wil be compensated bij the proportional increased lightpath.
the volume you have to use to be loaded in 96 well plate strongly depend on which kind of plate you use. Among round bottomed plate there are big differences. However I would not go under 50 uls, since the lower you go, the higher is the absorbance error in proportion to the volume error due to the loading process (pipetting error, retained liquid, drops on the walls, etc..). This unfavourable proportion increases if you are in the round part of the well.