2view™ double labeled secondary antibody

Innovative and unique detection method for Western blot

Expedeon’s 2view™ is an innovative and unique double labeled secondary antibody that enables extended detection within Western blotting.  The product consists of a unique ternary complex developed using our world leading InnovaCoat® and Lightning-Link® conjugation technology. 2view™ secondary antibodies are labeled simultaneously with InnovaCoat® GOLD nanoparticles and  horseradish peroxidase (HRP). The gold nanoparticles enable fast visible detection at nanogram level whereas the chemi- luminescent enzymatic detection enhances sensitivity down to picogram levels of protein.

Features & Benefits

  • Offers two modes of detection from a single antibody
  • Provides highly sensitive chemi-luminescence analysis
  • Easy to use with a single incubation
  • Fast visible detection
  • Dual level of sensitivity (nanogram and picogram level)
  • High signal-to-noise ratio

There are two levels of detection:

  1. Visible detection with InnovaCoat® GOLD
  2. Chemiluminescence via HRP & ECL Reagents (ECL Pico)

When performing a Western blot the amount of protein of interest is usually unknown; 2view™ solves this problem enabling two detections on the same blotted membrane: if the analyte is present down to nanogram level a red band will be visible; for lower amounts of analyte (picogram) the membrane can be developed using a chemiluminescent substrate (Expedeon’s LumiBlue ECL Pico Substrate).

 1st level of detection – 40nm InnovaCoat® GOLD

Molecular weight marker with visible 40nm Gold Nanoparticle bands

 Up to nanograms/single nanogram of analyte the detection relies on InnovaCoat® Gold nanoparticles and a red band will be visible by naked eye

2nd level of detection – HRP (ECL Pico)

Molecular weight marker with clear protein bands using Enhanced Chemiluminescence Pico

Down to picograms of analyte the detection relies on the activity of HRP after the addition of ECL Pico (Expedeon).  The signal can be recorded using a CCD camera or an X-Ray film

Comparison between 2view™ and traditional Western blot detection methods

Western blot flow chart including ECL detection and gold nanoparticle detection

Kit Contents:

  • 10ml 2view™ anti-Rabbit / anti-Mouse/ anti-Goat [Gold/HRP]

Related Products:

For more information about this product please don’t hesitate to get in touch.

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Sensitivity of gel and membrane staining methods

Figure 1. Transferrin (TF) was run on a 4-12% Bis-Tris gel (Expedeon, NBT41212), under non-reducing conditions for 40min at 180V in 1x MOPS (Expedeon, NXB75500). The gel was stained with InstantBlue™ (ISB1L) for one hour on the gel rocker (A). TF was blotted onto a nitrocellulose membrane (Expedeon, NXA19020), using 1x Tris-Glycine buffer (Expedeon, NXB86500) + 10% methanol. Membrane was reversibly stained with Ponceau S (B). After destaining, the membrane was blocked in 1x TBS, 0.05% Tween20 (Sigma, P1379-500ML), 5% BSA (VWR, 421501J) for 1 hour at RT on the gel rocker and then incubated O/N with anti-transferrin rabbit polyclonal diluted 1:5000 in 1x TBS, 0.05% Tween20 , 5% BSA on the gel rocker. Membrane was washed 3 times with 1x TBS, 0.05% Tween20 (5 minutes each wash on the gel rocker) and incubated for one hour with 2view™GAR [Gold/HRP] diluted 1:25 in blocking buffer (C).

 

Comparison of 2view™ GAR [Gold/HRP] and Goat anti-Rabbit HRP

Figure 2. Transferrin (TF) was run on a 4-12% Bis-Tris gel (Expedeon, NBT41212), under non-reducing conditions for 40’ at 180V in 1x MOPS (Expedeon, NXB75500). TF was blotted onto a nitrocellulose membranes (Expdeon, NXA19020), using 1x Tris-Glycine buffer (Expedeon, NXB86500) + 10% methanol. Membranes were blocked in 1x TBS, 0.05% Tween20 (Sigma, P1379-500ML), 5% BSA (VWR, 421501J) for 1 hour at RT on the gel rocker. Membranes were incubated O/N with anti-transferrin rabbit polyclonal diluted 1:5000 in 1x TBS, 0.05% Tween20 , 5% BSA on the gel rocker. Membranes were washed 3 times with 1x TBS, 0.05% Tween20 (5 minutes each wash on the gel rocker) and incubated for one hour with 2view™GAR [Gold/HRP] diluted 1:25 in blocking buffer and with Goat anti-Rabbit-HRP diluted 1:25.000 in blocking buffer on the gel rocker. Membranes were washed 3 times with 1x TBS, 0.05% Tween20 (5 minutes each wash on the gel rocker) and developed with ECL Pico (Expedeon, ECLP0250). Exposure time: 3s, 10s, 30s, 60s.

1. Is 2view™ suitable for both nitrocellulose and PVDF membranes?
Yes. 2view™ has been tested on nitrocellulose and PVDF membranes from different manufacturers (Expedeon, NXA19020 and NXA29320 ; Invitrogen, LC2001; Biorad, 1620117; Merck Millipore, IPVH00010) and data showed reproducible results as well as high signal-to-noise ratio.

2. Is 2view™ suitable with different blocking agents other than BSA?
Yes. 2view™ is suitable with the most commonly used blocking agents, such as non-fat dry milk (3-5%), casein (1%) and fish skin gelatin (1%). Also, it is compatible with 0.15-0.5M NaCl and 0.05-1% Tween20®. Non-fat dry milk and casein are not recommended when using a biotin-avidin interaction as milk contains large and variable amounts of biotin.

3. What is the recommended dilution range for the primary antibody?
Primary antibody dilution range is often between 1:500 to 1:5000 dilution from a 1 mg/mL stock. However, this may not be true for all antibodies and you should also consider the range suggested in the supplier datasheet.

4. What is the recommended dilution range for 2view?
2view™ has been optimized for a 1:25 dilution. However, 2view™ will work in a dilution range from 1:10 to 1:50 but this could require further wash steps and longer incubation time, respectively.

5. Can diluted 2view™ in blocking buffer be re-used?
Yes, it can be re-used up to 3 times; store at +4°C for up to 2 weeks. It is recommended not to re-use 2view™ on a membrane probed with a different primary antibody.

6. Can other LumiBlue ECL solutions from Expedeon (i.e. ECL Extended, ECL Extreme) be used instead of ECL Pico to increase sensitivity?
Yes, however the primary antibody concentration must be optimized (higher dilution) to keep the background low.

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