Q1. How long is the recommended reaction time of luminescence solution and membrane?
A1. The reaction time is a few seconds. A longer reaction time, e.g., about 5 minutes, can be used without any problem.
Q2. How long is the recommended exposure time?
A2. The optimum exposure time varies according to several factors such as the quantity of the sample applied and the dilution of the secondary
antibody. If this is your first time, we recommend that you try employing 1, 10 or 30 seconds as the exposure time. When changing the luminescence
reagent from the one you are currently using, please refer to Fig1. Exposure Time Data
Q3. What is the recommended dilution ratio of the labeled secondary antibody?
A3. The optimum dilution ratio varies according to several factors such as the quantity of the sample applied and the exposure time.
When changing the luminescence reagent from the one you are currently using, please refer to Fig 2. Dilution Ratio of Labeled
Secondary Antibody Data
Q4. How long does the luminescence last?
A4. It last more than 24 hours after the reaction. However, the amount of luminescence gradually decreases. Please refer to
Fig 3. Luminescence Duration Data
Q5. Are the signals detected quantitatively?
A5. The quantitativity is confirmed not only in the high concentration range but also in the low concentration range.
Q6. The background was too high following detection under the same conditions that had been employed for the previously used luminescence reagent.
How do I deal with this problem?
A6. First, we recommend shortening the exposure time. The time is shortened to approximately 1/30-1/60 and 1-1/2 if the reagent was changed from
the commercially available high-sensitive long-lasting types
(Note 1) and the super-sensitive types (Note 2), respectively.
If the results are not improved despite shortening exposure time, the dilution ratio of the labeled secondary antibody should be changed.
If the reagent was changed from the commercially available high-sensitive long-lasting types, further dilute 4-10 fold.
If it was changed from the super-sensitive types, there is no need for a change. Please refer to Q2 and Q3.
Q7. The signals were detected, but the background is high. How do I lower the background and improve the SN ratio?
A7. The excess of luminescence solution may be left on the membrane after the reaction between the solution and membrane.
We recommend that the detection is carried out after the excess of the luminescence solution is fully removed.
Since this product exhibits strong luminescence in a short time, high SN ratio can be obtained by stopping the exposure while background is low.
Q8. How long are the reagents stable?
A8. ImmunoStar® LD has superior storage stability and can be used after more than 18 months from the date of manufacture.
However, since the luminescence reaction progresses upon the mixture of luminescence solution A and B, the mixture should be prepared before use.
Q9. Can fluorescence be detected?
A9. Unfortunately, fluorescent can not be detected.
(Note1) Classification of common luminescence reagents
Fig1. Exposure Time Data
… (Note 1) Change from high-sensitive long-lasting types … Shortening the exposure
time to 1/30 ~ 1/60 is recommended.
… (Note 2) Change from super-sensitive type … Employing the same exposure time or
shortening the time to 1/2 is recommended.
* If the sensitivity is poor, the signals can be detected with high sensitivity through longer exposure time.
(Data was provided by Dr. Tomita and Osawa, Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical
Sciences, The University of Tokyo)
Fig 2. Dilution Ratio of Labeled Secondary Antibody Data
… (Note 1) Change from high-sensitive long-lasting types … Further 4~10 fold dilution
from previously used ratio is recommended.
… (Note 2) Change from super-sensitive types … No need for change of the dilution ratio
of the labeled secondary antibody.
Fig3. Luminescence Duration Data
POD-conjugated mouse IgG to Rabbit IgG (DAKO #P-0260) was diluted 80000-fold. 1 µL of this solution was added on the fluorescence/luminescence
plate (Corning #3915) and after the addition of each 50 µL of the luminescence reagents, measurement was performed with a luminometer
(TECAN Ultra Evolution).