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
Membrane PVDF, nitrocellulose
Detection method CCD imager, X-ray film
Reprobing Feasible
Features
  • Excellent luminescence stability
  • Excellent quantitativity
  • Most sensitive in the series

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

  • 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

  • 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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