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Fluorescent Western Blot

Fluorescent Western blotting can offer many advantages and improve the highly robust Western blotting technique. Fluorescent Western blotting is a method that is becoming increasingly popular because it allows powerful quantitative detection of protein expression.

The basic principle behind the method is that the strength of fluorescence of a fluorophore bound to a detection antibody is proportional to the strength of protein expression. Since fluorescence does not occur enzymatically, as is the case with classical Western blotting, quantitation is much more reproducible.

Figure 1. Fluorescent Westernblotting: 1. The fluorophore-conjugated secondary recognizes the primary antibody bound to the protein of interest. 2. A light source is used to excite the fluorescent conjugate to fluorescence. 3. The excited dye emits light of a specific wavelength, which can be visualized by digital imaging. Thus, the protein of interest can be detected quantitatively.

Fluorescence vs Chemiluminescence Western Blotting

Chemiluminescent detection Fluorescent detection
Signal source Signal from enzymatic reaction Signal from fluorophore
Detection Photographic film, imaging instrument Imaging instrument with light source and filters
Sensitivity Good, wide variety of substrates available Good, wide variety of fluorophores available
Quantitation Single channel, Challenging normalization Multiple channel, easier normalization
Signal duration Minutes to hours Weeks to months

Blocking Buffer for Fluorescent Western blotting

Blocking Buffer for Fluorescent Western Blotting
  • Designed for western blotting using fluorochrome conjugated antibodies.
  • Consists of a proprietary protein formulation in TRIS buffered saline at pH 7.6 with thimerosal.
  • Ultra pure reagents dissolved in pharmaceutical grade water.
Fluorescent Westernblotting Blocking Buffer ABIN925618
Blocking Buffer for Fluorescent Western Blotting
  • Western Blot Blocking Buffer is ideal for infrared Western Blotting.
  • This product is a 2X concentrated stock solution. Prepare a 1X working solution by diluting 1 part 2X concentrate with 1 parts TBS or equivalent.
  • Filtered through a Millipore 0.22 micron filter.
Fluorescent Westernblotting Blocking Buffer ABIN6953293

Multiplex Duo Fluorescent Western Blotting Kits

Multiplex Duo fluorescent Western blotting kits are suited for simultaneous detection and quantification of specific protein populations in a biological sample. Using a combination of two antibodies selected for minimal cross reactivity, fluorescent detection method enables simultaneous quantitative analysis of multiple proteins within the same sample on the same blot.

Figure 2. Multiplex Duo Fluorescent Western Blotting Kits: Simultaneous detection of α-tubulin and GFP on a single blot using labeled secondary antibody conjugates. Probing of cell lysates and GFP with mouse anti-α-tubulin and chicken anti-GFP antibodies followed by 649 goat anti-mouse IgG (pseudocolored green) and 800 goat anti-chicken IgG (red) conjugates, and then imaged using Syngene G:BOX Imaging System resulted in comparable patterns of detection.

Name / Product link Fluorescence channels Reactivity
DyLight™ Multiplex 488/800 Duo Western Blot Kit 488 / 800 nm Mouse, Rabbit
DyLight™ Multiplex 549/800 Duo Western Blot Kit 549 / 800 nm Mouse, Rabbit
DyLight™ Multiplex 649/488 Duo Western Blot Kit 649 / 488 nm Mouse, Rabbit
DyLight™ Multiplex 649/549 Duo Western Blot Kit 649 / 549 nm Mouse, Rabbit
DyLight™ Multiplex 649/800 Duo Western Blot Kit 649 / 800 nm Mouse, Rabbit

Multiplex Trio Fluorescent Western Blot Kits

The multiplex Trio fluorescent Western blot kit is suited for simultaneous detection and quantification of specific protein populations in a biological sample. Using a combination of three antibodies selected for minimal cross reactivity, fluorescent detection method enables simultaneous quantitative analysis of multiple proteins within the same sample on the same blot.

Name / Product link Fluorescence channels Reactivity
DyLight™ Multiplex 649/488/800 Trio Western Blot Kit 649 / 488 / 800 nm Chicken, Mouse, Rabbit
DyLight™ Multiplex 649/549/800 Trio Western Blot Kit 649 / 549 / 800 nm Chicken, Mouse, Rabbit

DyLight™ conjugated Secondary Antibodies

Product
Reactivity
Clonality
Application
Cat. No.
Quantity
Datasheet
Reactivity Rabbit
Clonality Polyclonal
Application WB, FLISA, FM
Cat. No. ABIN6699122
Quantity 100 μg
Datasheet Datasheet
Reactivity Mouse
Clonality Polyclonal
Application WB, FLISA, FM
Cat. No. ABIN6699038
Quantity 100 μg
Datasheet Datasheet
Reactivity Mouse
Clonality Polyclonal
Application WB, FLISA, FM
Cat. No. ABIN6699028
Quantity 100 μg
Datasheet Datasheet
Reactivity Rabbit
Clonality Polyclonal
Application WB, FLISA, FM
Cat. No. ABIN6699113
Quantity 100 μg
Datasheet Datasheet

TrueBlot® HRP-conjugated Secondary Antibodies

Product
Reactivity
Clonality
Application
Cat. No.
Quantity
Datasheet
Reactivity Mouse
Clonality Monoclonal
Application IP, WB
Cat. No. ABIN1589977
Quantity 200 μL
Datasheet Datasheet
Reactivity Mouse
Clonality
Application FLISA, FACS, FM, IHC, WB
Cat. No. ABIN6698836
Quantity 100 μL
Datasheet Datasheet
Reactivity Goat
Clonality Monoclonal
Application IP, WB
Cat. No. ABIN1589970
Quantity 200 μL
Datasheet Datasheet
Reactivity Sheep
Clonality Monoclonal
Application IP, WB
Cat. No. ABIN1589972
Quantity 200 μL
Datasheet Datasheet
Reactivity Mouse
Clonality Monoclonal
Application IP, WB
Cat. No. ABIN1589968
Quantity 100 μg
Datasheet Datasheet

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