WAKODistributors WAKO Wako USA WAKO Wako GmbH WAKO Contact Us

Online Catalog | Product Information | Ordering Information | MSDS | Certification of Analysis | Catalog Request | Journals |

Laboratory Chemicals Division > Product Information > I > ImmunoStar® LD

ImmunoStar® LD

Wako Catalog No. 296-69901 (  200 cm2)
                                292-69903 (1000 cm2)
                                290-69904 (2000 cm2)

Immunoblot Assay Procedure

FAQ

ImmunoStar® LD from Wako are designed for a simple and highly sensitive immunoblotting, utilizing detection by enhanced chemiluminescence using a unique luminol derivative L-012 as the substrate.

Features:

  1. High sensitivity (~10-14; femto gram level    Note 1 )
  2. Long term duration of chemiluminescence (CHL) (usually up to 24 hours)  Fig 3
  3. Low background (a high ratio of S/N)
  4. Stability of the product (up to 18 months)

ImmunoStar® LD
Wako Catalog No. (Pkg Size)
296-69901 (200 cm2)
292-69903 (1000 cm2)
290-69904 (2000 cm2)
Kit Contents
Luminescence Solution A
10 mL
50 mL
100 mL
Luminescence Solution B
10 mL
50 mL
100 mL


Luminescent Principle of ImmunoStar® LD system


 

 
Procedure Summary                           FAQ
Immunoblot Assay Procedure-1
  1. Prepare a protein blotted membrane, 100 cm2, by electrophoretic transfer or dot blotting
  2. Introduce the membrane into 50 mL of Blocking Buffer*1, and incubate for 1 hour at room temperature (RT) or incubate overnight at a cool place.
  3. Wash the membrane with 50 mL of Wash Solution*2 for 15 minutes, followed by the 5 minute washing, twice.
  4. Prepare primary antibody with Antibody Diluent*3 in an appropriate concentration and incubate the membrane with the antibody solution for 1 hour at RT.
  5. Wash the membrane with 50 mL of Wash Solution*2 for 15 minutes, followed by the 5 minute washing, twice.
  6. Prepare secondary antibody with Antibody Diluent*3 in an appropriate concentration and incubate the membrane with the antibody solution for 1 hour at RT. FAQ
  7. Wash the membrane with 50 mL of Wash Solution*2 for 15 minutes, followed by the 5 minute washing, twice.



(*1) Blocking Buffer
    (5% Skim milk/PBS-0.1% Tween 20)

  •                  Skim milk                           5 g
                     10% NaN3                           1 mL

            Prepare the 100 mL solution by adding PBS-0.1% Tween 20.
       (Note)
           Prepare just before use.

    (*2) Wash Solution
        (Select Wash Soln. A or B depending on your purpose)

  • Wash Solution A: PBS-0.1% Tween 20
                     NaCl                           8.0 g
                     KCl                            0.2 g
                     Na2HPO4 12H2O     2.9 g
                     KH2PO4                   0.2 g
                     Tween 20                   1.0 g
               Prepare the 1000 mL solution by adding H2O
       (Note)
         Adjust the pH to 7.4
         Stable at room temperature.
         Preparation of the 10x Stock Wash Solution is recommended.

  •     Wash Solution B: TBS-0.1% Tween 20
                     Tris base                 3.0 g
                     NaCl                      8.0 g
                     KCl                        0.2 g
                     Tween 20               1.0 g
               Prepare the 1000 mL solution by adding H2O
       (Note)
         Adjust the pH to 7.4
         Stable at room temperature.
         Preparation of the 10x Stock Wash Solution is recommended.

    (*3) Preparation of Antibody Diluent
        (Select Antibody Diluent A or B depending on your purpose)

  • Antibody Diluent A: 0.1% BSA/PBS-0.1% Tween 20
                      BSA                    0.1 g

         Prepate the 100 mL soln. by adding PBS-0.1% Tween 20

  • Antibody Diluent B: 0.1% BSA/TBS-0.1% Tween 20
                      BSA                    0.1 g

         Prepate the 100 mL solution by adding TBS-0.1% Tween 20

  • Immunoblot Assay Procedure-2
    1. Preparation of Working Solution;           FAQ
      Just before use, prepare the Luminescence Working Solution by mixing Luminescence Solution A and Luminescence Solution B at a ratio of 1:1

    2. Add the Working Solution onto the wrap by pipette.  The volumes of it needs for 100 µL/cm2.



    3. Place the blot protein side down with the membrane on the wrap.



    4. Incubate the blot for a few seconds to 5 minutes.



    5. Drain the excess Working Solution.



    6. Cover the blot with a clean wrap and remove any air bubbles.










    7. Expose the wrapped membrane to X-ray film or image analyzer for a few seconds to 1 minute.             FAQ


    Related Product

    <LOW-SENSITIVE TYPE    Note 1>
    ImmunoStar® Reagents              Package Insert
    Wako Catalog No. (Pkg Size)
    295-55201 (1000 cm2)
    291-52203 (5000 cm2)
    Kit Contents
    Luminescent Solution A
    70 mL
    330 mL
    Luminescent Solution B
    70 mL
    330 mL
    Luminescent Solution C
    30 mL
    120 mL
    Frequently Asked Questions on ImmunoStar® LD


    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

    Fig/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.

    Fig/Exposure time data 1

    (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.

    Fig/Exposure time data 2

    (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.

    Fig/Exposure time data 3

    (Note 2) Change from super-sensitive types No need for change of the dilution ratio of the labeled secondary antibody.

    Fig3. Luminescence Duration Data

    (Method)
    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).

    Fig/Luminescence Duration Data

     

     


    Copyright© Wako Pure Chemical Industries, Ltd.  All Rights Reserved.