I am trying to detect my his-tagged protein (dilutions up to 1 pg/ml) by anti his antibody coupled with HRP. I am planning to detect by luminol based chemiluminescence (by adding HRP substrates). I'm using black flat bottom (transparent) 96 well plate for this purpose. Two different antibody dilutions (1:1000, 1:2000) used. Blocking with 5%BSA +0.1% Tween 20 in PBS. I considered the blank well with only substrates and without antigens. Blank wells were also incubated with antibody but washed with PBS in successive preparation. I'm using Teacan pro 200 for detection of the absorbance at 425 nm. I was expecting decrease of signal with antigen dilutions and the blanks will give signal accordingly very low. But I've relatively high signal with blanks. Is it a problem with washing? Or should I avoid incubation of blank well with antibody? Any suggestion with the protocol?
It is unclear from your description whether the blank wells were also blocked with BSA/Tween. Do they? If you didn't, it is possible that the antibody stick to the empty well just in a non-specific manner. Background is also reduced by including tween20 (0.1% v/v) in all washes
1% BSA with 0.1% Tween 20 would be good as blocking agent. This may reduce the back ground also.
Which buffer you are using for washing? You can use PBS with 0.1% Tween 20 for washing to decrease the back ground.
A typical 3 times wash step with PBS containing 0.1% Tween 20 between each step of the ELISA would help remove any unbound material and a final 3 times wash with PBS containing 0.1% Tween 20 and a fourth wash with just PBS prior to adding your substrate will decrease the background and any intereference. This is because tween, a detergent can also interfere with your substrate and make results inconsistent.
Thanks All for the feedback. And did anyone tried to measure the chemiluminescence (HRP substrates) in plate reader? I do washing with PBS+0.1 % Tween 20 each time in washing steps. I will try with only PBS wash as Oluseyi suggested. Thanks.
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