Microplate reader : negative value in Blank - (Dec/05/2007 )

hi all

i do the protein quantitation for my protein sample using micro-BCA assay and measured using microplate reader with wavelength 570nm.. The result look ok, except the blank give me a negative reading...

should i redo my experiment? any solution for this, because i do not have many protein to spend for the quantitation dy.. any method can be used to normalised the negative reading in the blank ??

thanz ya

Hi

i think there is a problem with the reader. but if the neg value is close to 0 so it might be ok.

what value do u get from emty well in the plate compared to the blank, it have to be smaller.


bye

with what did you zero the reader?

you should set the blank to zero (zero the reader with the blank). if you don't want to or can't reread the plate then you can subtract the blank from your readings.

in the future zero the reader with the blank.

by the way, i would be more concerned if any of the samples read lower than the blank.

the microplate reader we use constantly gives a value for the blank (positive and negative). it's just a problem with the machine (most of the time).
simply subtract the blank from the other readings.
unless the number is really big, don't worry about it.

V

Thanz for the reply...

the blank value is about -0.020, -0.017 and -0.019... i read the plate for more than 20 times, and i even restart the microplate reader and take the reading again. but the blanks still give me these negative value (more and least the same)...

i do not have any value larger than the blank... all my sample well give me positive value, the empty well give me reading about 0.002, 0.003....

so if i subtract the blank value, that mean, the empty well give me a reading of 0.0023??

empty wells are not the best blanks. you need some solution (or water) in them to match the optical qualities of the wells with sample.

have you ever blanked a spectrophotometer with a empty cuvette then compare that to one filled with water? the empty cuvette reads higher than the filled one.

blanks contain the solution but not the stuff you're trying to measure.
ie, for protein, the blank would contain only the bradford stuff... no protein.
ie, for RNA, the blank would contain only water... no RNA.
you're not going to get anywhere by measureing an empty well.

V