WesternDot™ Fluorescent Immunodetection Kits
 | Bring your western blotting into the 21st centuryWesternDot™ 625 detection 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.
|
${comergentUrl}
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.
 |  | |
| 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. | ||
Procotol to Combine the WesternDot™ Technology with WesternBreeze® Chemiluminescent Detection
Simply by integrating WesternDot™ detection technology into your current WesternBreeze® chemiluminescent western blot workflow, two proteins of interest can be visualized on one blot at the same time with no additional solutions or steps.
Figure 4. Multiplexed WesternDot™ 625 and WesternBreeze® protein detection. A 2-fold dilution series (10 µg to ~160 ng) of untreated (left side of blots) and wortmannin-treated (right side) Jurkat cell extracts was separated on NuPAGE® Novex® 4–12% Bis-Tris gels and transferred to nitrocellulose membranes using the iBlot® dry blotting system. Blots were coincubated with rabbit anti-AKT and mouse anti-pAKT antibodies and detected with WesternDot™ reagents (left). The same blots were incubated with WesternBreeze® reagents to detect the alternate antigen (right). For Qdot® 625 detection, the membrane was imaged on a FujiFILM LAS-4000 imager with UV epi-illumination, a 605DF40 emission filter, and an exposure time of 20 sec. For CDP-Star® detection, the membrane was imaged without an illumination or emission filter, with an exposure time of 1 min.
Figure 4. Multiplexed WesternDot™ 625 and WesternBreeze® protein detection. A 2-fold dilution series (10 µg to ~160 ng) of untreated (left side of blots) and wortmannin-treated (right side) Jurkat cell extracts was separated on NuPAGE® Novex® 4–12% Bis-Tris gels and transferred to nitrocellulose membranes using the iBlot® dry blotting system. Blots were coincubated with rabbit anti-AKT and mouse anti-pAKT antibodies and detected with WesternDot™ reagents (left). The same blots were incubated with WesternBreeze® reagents to detect the alternate antigen (right). For Qdot® 625 detection, the membrane was imaged on a FujiFILM LAS-4000 imager with UV epi-illumination, a 605DF40 emission filter, and an exposure time of 20 sec. For CDP-Star® detection, the membrane was imaged without an illumination or emission filter, with an exposure time of 1 min.
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
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 | |
| Included |
| Included |
| Included |
| 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
| Invitrogen
|
Bio-Rad
| KODAK
|
FUJIFILM Life Science
| Syngene
|
GE Healthcare
| UVP
|
Spectral properties of the Qdot® 625 streptavidin conjugate
 | Figure 5—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.
Find out more about Qdot® nanocrystals at www.invitrogen.com/qdots.
