Peroxidase Luminescent Substrates
ImmunoStar® Series are chemiluminescence reagents for Western blot for detection of peroxidase (HRP; POD)-labeled antibodies. Two brands, Zeta and LD, are available according to the experimental system.。
Selection Guide for ImmunoStar® Series
| Product Name | ImmunoStar® Zeta | ImmunoStar® LD |
|---|---|---|
| Type | Balanced | Emphasis on luminescence intensity |
| Luminescence intensity(measure of sensitivity) | Highly sensitive 4- to 20-times higher luminescence intensity than the conventional products of FUJIFILM Wako |
Super-sensitive 20- to 100-times higher luminescence intensity than Zeta |
| luminescence stability | ◎ | △ |
| Cost | 〇 | 〇 |
| Membrane | PVDF, nitrocellulose | |
| Detection method | CCD imager, X-ray film | |
| Reprobing | Feasible | |
| Features |
|
|
ImmunoStar® Zeta
ImmunoStar® Zeta is a highly sensitive luminescence reagent with an emphasis on good balance of luminescence intensity, luminescence stability, and quantitative dynamic range. It provides stable luminescence for a long period of time while exhibiting a strong luminescence signal. It also generates highly linear calibration curves over a wide range of protein content.
Luminescence intensity and stability
FLAG-BAP was serially diluted to compare the luminescence intensity and stability of ImmunoStar® Zeta with those of Competitive product B (highly sensitive). ImmunoStar® Zeta exhibited a luminescence signal comparable to that of Competitive product B (highly sensitive) and provided more stable luminescence for a long period of time.
| Sample | ：FLAG-BAP |
| Gel | ：SuperSep™ Ace, 5-20%, 17 well (Product Number: 194-15021) Primary antibody: Anti-DYKDDDDK Tag, Monoclonal Antibody (Product Number: 014-22383) |
| Primary antibody | ：Anti-DYKDDDDK Tag, Monoclonal Antibody (Product Number: 014-22383) |
| Secondary antibody | ：Anti-Mouse IgG (H+L), Rabbit, IgG fraction, peroxidase binding (Product Number: 018-23643) |
| Exposure time | ：8 seconds (LAS4000, standard) |
| Lane No. | FLAG-BAP (ng) |
|---|---|
| 1 | 20 |
| 2 | 10 |
| 3 | 5 |
| 4 | 2.5 |
| 5 | 1.3 |
Quantitative dynamic range
FLAG-BAP was serially diluted to compare the quantitative dynamic range of ImmunoStar® Zeta with that of Competitive product B. ImmunoStar® Zeta generated a highly linear (R2 ≥0.99) calibration curve in the same protein content range as Competitive product B.
| Sample | ：FLAG-BAP |
| Gel | ：SuperSep™ Ace, 10-20%, 13 well (Product Number: 191-15031) |
| Membrane | ：ClearTrans® SP PVDF Membrane, Hydrophobic, 0.2 µm (Product Number: 033-22453) |
| Primary antibody | ：Anti-DYKDDDDK Tag, Monoclonal Antibody (Product Number: 014-22383), 1,000-fold diluted |
| Secondary antibody | ：Anti-Mouse IgG (H+L), Rabbit, IgG fraction, peroxidase binding (Product Number: 018-23643), 40,000-fold diluted |
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Lane No. FLAG-BAP (ng) 1 50 2 25 3 12.5 4 6.25 5 3.13 -
Lane No. FLAG-BAP (ng) 6 1.56 7 0.78 8 0.39 9 0.20 10 0.10
- For quantitative analysis, create a linear standard curve with a coefficient of determination of R2≥0.99, and use this curve if the luminescence intensity of test samples measured at the same time are within its range.
Comparison with other competitive products -Luminescence intensity and stability-
The luminescence intensity of ImmunoStar® Zeta is comparable to that of femtogram-level competitive products (Competitive products D and E). In addition, the luminescence intensity of ImmunoStar® Zeta at 90 minutes after the reaction is maintained at the same level as that of Competitive product D, which is characterized by persistent luminescence.
| Sample | ：FLAG-BAP |
| Gel | ：SuperSep™ Ace, 10-20%, 17 well (Product Number: 198-15041) |
| Primary antibody | ：Anti-DYKDDDDK Tag, Monoclonal Antibody (Product Number: 014-22383), 4,000-fold diluted |
| Secondary antibody | ：Anti-Mouse IgG (H+L), Rabbit, IgG fraction, peroxidase binding (Product Number: 018-23643), 20,000-fold diluted |
| Exposure time | ：60 seconds (LAS4000, standard) |
| Lane No. | FLAG-BAP (ng) |
|---|---|
| 1 | 20 |
| 2 | 10 |
| 3 | 5 |
| 4 | 2.5 |
| 5 | 1.25 |
ImmunoStar® LD
ImmunoStar® LD is a highly sensitive luminescence reagent with an emphasis on luminescence intensity. It is suitable for detecting minor proteins such as lowly expressed proteins.
Luminescence intensity
ImmunoStar® LD detected FLAG-BAP more sensitively than Competitive product B and ImmunoStar® Zeta.
| Sample | ：FLAG-BAP |
| Gel | ：SuperSep™ Ace, 10-20%, 17 well (Product Number: 198-15041) |
| Primary antibody | ：Anti-DYKDDDDK Tag, Monoclonal Antibody (Product Number: 014-22383), 2,000-fold diluted |
| Secondary antibody | ：Anti-Mouse IgG (H+L), Rabbit, IgG fraction, peroxidase binding(Product Number: 018-23643), 20,000-fold diluted |
| Exposure time | ：60 seconds (LAS4000, standard) |
| Lane No. | FLAG-BAP (ng) |
|---|---|
| 1 | 20 |
| 2 | 10 |
| 3 | 5 |
| 4 | 2.5 |
| 5 | 1.3 |
Luminescence stability
Anti-Mouse Immunoglobulins/HRP (DAKO, Product Number: P0260) was diluted 80,000 times. Then 1 μL of this solution was placed to a fluorescence luminescence plate (Corning Incorporated, Product Number: 3915), and 50 μL each of luminescent reagents was added for measurement with a luminometer (TECAN Ultra Evolution).
The luminescence intensity decreased by 61% at 120 minutes after the reaction compared with that at 5 minutes after the reaction.
Quantitative dynamic range
FLAG-BAP was serially diluted to determine the protein content range with a highly linear calibration curve. ImmunoStar® LD generated a highly linear (R2 ≥0.99) calibration curve in the protein content range of 0.39 to 6.25 ng.
Since ImmunoStar® LD has a very high luminescence intensity and therefore a high signal decay rate, the quantitative dynamic range is narrower than that of ImmunoStar® Zeta. ImmunoStar® Zeta is recommended for quantitative analysis of target antigens.
R2 ≥0.99 in the antigen range of 0.39 to 6.25 ng (Lane No. 1 to 5) (1.2-order)
| Sample | ：FLAG-BAP |
| Gel | ：SuperSep™ Ace, 10-20%, 13 well (Product Number: 191-15031) |
| Membrane | ：ClearTrans® SP PVDF Membrane, Hydrophobic, 0.2 µm (Product Number: 033-22453) |
| Primary antibody | ：Anti-DYKDDDDK Tag, Monoclonal Antibody (Product Number: 014-22383), 1,000-fold diluted |
| Secondary antibody | ：Anti-Mouse IgG (H+L), Rabbit, IgG fraction, peroxidase binding (Product Number: 018-23643), 40,000-fold diluted |
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Lane No. FLAG-BAP (ng) 1 6.25 2 3.13 3 1.56 4 0.78 5 0.39 6 0.20 7 0.10 -
- * For quantitative analysis, create a linear standard curve with a coefficient of determination of R2 ≥0.99, and use this curve if the luminescence intensity of test samples measured at the same time are within its range.
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