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Protein Expression and Analysis

WesternDot™ Western Blotting Kits

Bring your western blotting into the 21st century
WesternDot™ 625 kits allow direct imaging of blots while avoiding the need to spend time and money optimizing your detection on film.  Get results equivalent to routine ECL and stop multiple trips to the darkroom to process film.

  • Save time and hassle—No more frustrating ECL optimization
  • Reduce costs—Eliminate the need for X-ray films and developer
  • Ready-to-go—Works with existing gel imagers or UV light boxes

Get ultrasensitive detection from your western blots

The WesternDot™ 625 Western Blot Kits offer the unique properties of our Qdot® 625 nanocrystals combined with the high-affinity streptavidin-biotin binding reaction to give you straightforward, high-sensitivity detection of proteins on western blots. Incorporating a standard western blotting protocol, the detection step employs a biotinylated secondary antibody followed by the key component, Qdot® 625 streptavidin conjugate.
Workflow diagram for the WesternDot™
Figure 1—Workflow diagram for the WesternDot™ 625 Western Blotting Kit.
Example of WesternDot™ 625 Western Blotting Kit

Example of Standard ECL
Figure 2 & 3—Example of results using the WesternDot™ 625 Western Blotting Kit. Total proteins (2-fold dilution series ranging from 10 μg to ~10 ng) from Jurkat cell extract were analyzed on a NuPAGE® Novex® 4–12% Bis-Tris gel and then transferred to an Immobilon™-FL PVDF membrane. Immunodetection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and endogenous “housekeeping” protein in the Jurkat cell extract, was performed with the WesternDot™ 625 Goat Anti-Mouse Western Blot Kit using a mouse monoclonal anti-GAPDH antibody (Invitrogen Cat. no. 39-8600) at 1 μg/ml. The wet membrane was imaged using an Alpha Innotech HD2 instrument with a SYPRO® Red emission filter (620 +/– 40 nm) and excitation at 302 nm transillumination with an exposure time of 300 milliseconds.

Reduce costs and simplify your Western Blotting

Experience the ease of use and cost savings of our new WesternDot™ 625 kits: 

Lower cost for equivalent sensitivity
  • No additional reagents to buy—Everything you need is included in each kit.
  • Eliminate the waste of test film optimization—Does not require film for detection.
  • No additional buffers to prepare—All buffers and blocking solutions provided.

Product WesternDot™ 625 Western Blot Kit
Number of Reactions
20 mini-blots
Kit Contents
  • Secondary Antibody
 Included
  • Wash Buffer
 Included
  • Blocking Buffer
 Included
  • Staining Dishes
 Included
Enzyme Substrate Not needed
Dark Room
Not needed
Autoradiography Films
Not needed
Autoradiography Film processing reagents
Not needed
Signal Stability
Weeks to months
Sensitivity
Low picogram/band
Membrane compatibility

Nitrocellulose, PVDF

Use WesternDot™ reagents with many imaging instruments

The table below lists the instruments that are compatible with the WesternDot™ 625 kit reagents. In addition, any ethidium bromide imager or UV transilluminator equipped with a long-pass orange or orange/red gel filter will also provide acceptable results.

Imaging Instruments
Alpha Innotech
  • AlphaDigiDoc® PRO
  • AlphaImager® EP
  • AlphaImager® HP
  • FluorChem® HD2
Invitrogen
  • Safe Imager™ Blue-Light Transilluminator
Bio-Rad
  • ChemiDoc™ XRS
  • Gel Doc™ XR
  • PharosFX™ Systems
  • VersaDoc™ MP 4000
KODAK
  • Gel Logic
  • Image Station 4000 MM and PRO
  • Image Station 4000R and PRO
FUJIFILM Life Science
  • FLA-7000
  • FLA-8000
  • FLA-9000
  • LAS-3000
  • LAS-4000
Syngene
  • DigiGenius
  • G:Box Chemi
  • InGenius
  • ChemiGenius
  • U:Genius
GE Healthcare
  • Typhoon™
  • Storm™ (with Blue LED)
  • Visi-Blue™
UVP
  • Benchtop UV Transilluminators
  • BioDoc-It™
  • DigiDoc-It™
  • EC3™
  • FirstLight®

Spectral properties of the Qdot® 625 streptavidin conjugate



Figure 4—Excitation should be with deep blue or UV light in either trans- or epi-illumination modes. Lasers with 473 nm or 488 nm excitation are acceptable. Emission filters that are qualified for ethidium bromide, SYPRO® Ruby, SYPRO® Red, Qdot® 605, Qdot® 625, or Qdot® 655 will be suitable. Some instrument manufacturers produce filters specific for Qdot® nanocrystals; for those, contact the manufacturer.

Qdot® nanocrystals deliver superior brightness and photostability

The remarkable photostability of Qdot® nanocrystals enables long-term imaging experiments under conditions that would lead to the photoinduced deterioration of other types of fluorophores. With their broad excitation and narrow, symmetric emission properties, the Qdot® nanocrystals require only a single excitation source (typically <450 nm), facilitating multiplex analysis of multiple targets or events in a single sample.

Unique structural properties translate to powerful fluorophore performance

Fundamentally, Qdot® nanocrystals are fluorophores. The Qdot® products combine the revolutionary fluorescence performance inherent in the nanocrystal structure with a highly customizable surface for directing their bioactivity, producing a fluorescent probe that outperforms traditional dyes in many fluorescence applications.

Find out more about Qdot® nanocrystals at www.invitrogen.com/qdots. 
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