Stunningly easy western blot imaging from start to finish

Capture images and analyze data from your western blots and gels efficiently and easily using the Invitrogen iBright Imaging Systems. These high-performance instruments enhance the western blotting experience through advanced automated features and an interface that is easy to use for researchers of all experience levels. Be empowered to take on your research challenges and discover western blot imaging designed to work for you.

“The iBright Imager is incredibly user friendly!”

– Rebecca Sinnott DeVaux, SUNY

Open the tabs below to learn more about how our iBright Imaging Systems will help streamline your western blot and gel imaging experience.

Feel right at home—The experience begins with our capacitive LCD touch screen, which responds to inputs like other popular touch screen devices do.

Figure 1. Touch screen controls.

Load and go—Insert samples using the motorized drawer. The interface has a simple, logical layout of functions and features, making iBright Imaging Systems easy to use with minimal training. Workflows are similar between imaging modes—for a smooth imaging experience regardless of sample type. 

Hassle-free sample alignment, focus, and zoom—rather than having to open the sample door and repeatedly reposition your sample to achieve proper alignment, iBright Imaging Systems automatically determine the sample position and can rotate samples left or right up to 10° on a mechanically rotating sample stage. Mechanical rotation is preferable to digital rotation, as digital rotation can lead to data alterations. 

The iBright Imaging Systems automatically determine if the sample requires zoom in order to maximally utilize the field of view. If imaging a single blot, the camera will move toward the sample (up to 2x zoom). This puts the camera physically closer to the sample, which further maximizes sensitivity, as the light travels a shorter distance to the camera sensor. The iBright Imaging Systems automatically adjust focus for each level of zoom, producing crystal-clear images. 

Figure 3. Sample rotation. Graphic depicting iBright Imaging System stage before and after rotation.
Fig. 4. Digital vs mechanical sample rotation. Left: digital rotation (10°)—pixels rotate with digital rotation, so bands appear jagged. Right: Mechanical rotation (10°)—sample rotates so bands remain smooth as pixels remain aligned
Figure 5. Zoom function. At 1x zoom, the field of view is 22.5 cm (W) x 18.0 cm (H). Four full-sized mini blots or gels can be accommodated (left panel). The same blot outlined in the left panel, when repositioned in the center of the field of view, at 2x zoom (right panel).

Analysis in a fraction of the time—iBright Imaging Systems feature automatic on-board data analysis, which allows for instantaneous lane and band identification and molecular weight marker overlay, greatly simplifying and streamlining basic post-image data analysis. Quantitation and densitometry analysis can be performed directly on the instrument. Up to 4 blots or gels can be analyzed simultaneously, greatly increasing throughput.

Figure 6. Automatic lane and band identification of four mini blots in chemiluminescent blot acquisition mode.

Capture crystal-clear images—Smart Exposure technology rapidly determines optimal exposure time, which helps minimize the potential for over- or underexposed images, and the need to repeat exposures to get the desired signal. Combining Smart Exposure technology with a sensitive 9.1 megapixel (MP) cooled CCD camera, iBright instruments provide powerful imaging potential, which helps enable the detection of subtle differences in protein expression.

Figure 7. Detect subtle protein modifications using iBright Imaging Systems. HeLa cells were stimulated with IFN-alpha to induce protein phosphorylation, or with staurosporine to induce protein cleavage. Following stimulation, cells were lysed with IP Lysis Buffer (Cat. No. 87787). Equal amounts (20 µg) of the cell lysates were loaded onto 4–20% Tris-glycine gels (Cat. No. WT4202BOX). Proteins were transferred to nitrocellulose membranes (Cat. No. 88018) using the Thermo Scientific Pierce Power Blotter (Cat. No. 22834), and the membranes were then blocked with 5% milk in TBS-Tween 20 (TBST, Cat. No. 28360) for at least 30 min. The membranes were probed overnight at 4°C with antibodies against PARP (Cat. No. MA5-15031, 1:1,000 dilution) (left panel), or phospho-STAT3 (Cat. No. MA5-15193, 1:500 dilution) and STAT3 (Cat. No. MA1-13042, 1:5,000 dilution) (right panel). Blots were washed 3 times for at least 10 min each in TBST, and probed with HRP-conjugated goat anti-rabbit secondary antibody (Cat. No. 31460, 20 ng/mL) (left panel) or HRP-conjugated goat anti-mouse secondary antibody (Cat. No. 31430, 20 ng/mL) (right panel) for 1 hr at room temperature. Blots were again washed 3 times for at least 10 min each in TBST, and developed using SuperSignal West Dura substrate (Cat. No. 34076). Bands were visualized on the iBright FL1000 Imaging System with 2 min exposure (PARP), 3 min exposure (phospho-STAT3) and 52 sec exposure (STAT3).

Figure 8. iBright Imaging Systems feature a powerful 9.1 MP camera for greater sensitivity compared to instruments with a lower-resolution camera. Two-fold serial dilutions of HeLa cell lysate (starting at 80 µg/lane) were loaded onto 4–20% Tris-glycine gels (Cat. No. WT4202BOX). Proteins were transferred to nitrocellulose membranes (Cat. No. 88018) using the Pierce Power Blotter (Cat. No. 22834), and the membranes were then blocked with 5% milk in TBS-Tween 20 (TBST, Cat. No. 28360) for at least 30 min. The membranes were probed overnight at 4°C with antibodies against Ku80 (Cat. No. MA5-14953, 1:1,000 dilution) or DDX3 (Cat. No. PA5-17165, 1:1,000 dilution). Blots were washed 3 times for at least 10 min each in TBST, and probed with an HRP-conjugated goat anti-rabbit secondary antibody (Cat. No. 31460, 10 ng/mL (Ku80 blot) and 40 ng/mL (DDX3 blot)) for 1 hr at room temperature. Blots were again washed 3 times for at least 10 min each in TBST, and developed using SuperSignal West Pico PLUS substrate (Cat. No. 34580). Bands were visualized on the iBright FL1000 Imaging System (top blots), another imaging device with a lower-quality CCD camera (bottom blots), each with 10 sec exposures.

Figure 9. Comparison of detection sensitivity and dynamic range for film and the iBright FL1000 Imaging System. A luminometer reference plate emitting light at varying fixed intensities and specific wavelength (540nm) was used to expose film or to acquire an image on the iBright FL1000 Imaging System for 10 sec (left panel). More luminometer spots are visible on the image from the iBright instrument, indicating higher sensitivity compared to film. iBright and film 1 min exposures to the same luminometer plate (middle panel) with graphical analysis. Analysis reveals the better signal linearity and dynamic range of signals acquired using the iBright FL1000 Imaging System compared to film (right panel).

Big field of view, small footprint—The iBright Imaging Systems feature a light path design that  helps enable a large functional viewing area in a relatively small instrument footprint.

Figure 10. The large field of view (22.5 x 18.0 cm) enables capture of up to 4 mini blots or gels. Left: four mini blots. Right: signals captured in fluorescent blot mode.

Long-life epi-LED illumination—The iBright FL1000 Imaging System uses a simple combination of two high-quality long-life epi-LEDs for fluorescent imaging applications. One broad spectrum white LED is used as the light source for RGB fluorescence and far-red fluorescence. The other LED is optimized for near-IR fluorescence. Light from these sources passes through our excitation and emission filters to enable many possible reagent options for protein gel, nucleic acid gel, and blot imaging applications (Table 1).

Table 1. Overview of the types of data that can be captured from gels and blots.

Imaging capability What kind of signal can be captured?
Protein gel Colorimetric staining of gels (e.g., Coomassie dye, silver) and membranes (e.g., Ponceau S, Thermo Scientific MemCode stain)
Nucleic acid gel Ethidium bromide and Invitrogen SYBR dye staining
Chemiluminescent blot Chemiluminescence using all popular HRP and AP substrates (e.g., Thermo Scientific SuperSignal and Invitrogen WesternBreeze substrates)
Fluorescent blot* Fluorescence with popular RGB (visible range) and near-IR fluorophores (e.g., Invitrogen Alexa Fluor, Alexa Fluor Plus, Thermo Scientific DyLight dyes)
*FL1000 model only.

Accelerate your research—The iBright FL1000 model features five fluorescence channels, permitting up to 4-color fluorescent western blot multiplexing and expanding your possibilities for studying multiple proteins in a single blot. Obtain meaningful and representative comparisons to enhance your experiments. Smart Exposure technology further improves acquisition of multiplex fluorescent western blot data, because signal-to-noise ratios are optimized for each fluorescent channel separately.

Figure 11. Two-channel (near-IR) imaging of a fluorescent western blot. Several HA-tagged proteins were expressed in HeLa cell extract using the Thermo Scientific 1-Step Human High-Yield Mini IVT Kit (Cat. No. 88890) and appropriate expression-ready clones. The resulting reaction mixtures were prepared for SDS-PAGE and electrophoresed: Erythropoietin precursor, lanes 1-2; Streptokinase, lanes 3-4; Caveolin-1, lanes 5-6; molecular weight marker, lane 7; Argonaute-2, lanes 8-9; Regulatory-associated protein of mTOR, lanes 10-11; Bcl2-associated agonist of cell death, lanes 12-13; Retinoblastoma susceptibility protein, lanes 14-15. The proteins were transferred to a PVDF membrane using the Pierce Power Blotter (Cat. No. 22834), and the membrane was blocked and probed with the following primary antibodies: mouse anti-HA (Cat. No. 26183) and rabbit anti–cyclophilin B (Cat. No. PA1-027A). Cyclophilin-B is present in the HeLa cell extract and serves as a control in this experiment. The membrane was washed and probed with the following secondary antibodies in TBS-Tween 20: goat anti-rabbit Alexa Fluor Plus 800 (Cat. No. A32735) (pseudocolored in red) and goat anti-mouse Alexa Fluor Plus 680 (Cat. No. A32729) (pseudocolored in green). The membrane was washed and imaged on the iBright FL1000 Imaging System.
Figure 12. Four-channel imaging of a fluorescent western blot. Up to four different proteins can be imaged simultaneously on the same blot. HA-tagged RB-1 was expressed in HeLa cell extract using the 1-Step Human High-Yield Mini IVT Kit (Cat. No. 88890) and appropriate expression-ready clones. The resulting reaction mixture was prepared for reducing SDS-PAGE, serially diluted, and electrophoresed on a Novex WedgeWell 4–20% Tris-glycine gel (Cat. No. XP04200PK2). The protein was transferred to a PVDF membrane using the Pierce Power Blotter (Cat. No. 22834), and the membrane was blocked and probed with the following primary antibodies: chicken anti-calreticulin (Cat. No. PA1-903), rabbit anti-HSP90 (Cat. No. PA3-013), and mouse anti-p23 (Cat. No. MA3-414). The membrane was washed and probed with the following secondary antibodies in TBS-Tween 20: goat anti-chicken Alexa Fluor 546 (Cat. No. A11040) (pseudocolored in yellow), goat anti-rabbit Alexa Fluor Plus 800 (Cat. No. A32735) (pseudocolored in green), and goat anti-mouse Alexa Fluor Plus 680 (Cat. No. A32729) (pseudocolored in red). The membrane was again washed and probed for 1 hr with mouse anti-HA primary antibody directly conjugated to Alexa Fluor 488 (Cat. No. 26183-D488) (pseudocolored in blue), in TBS-Tween 20. The membrane was washed and imaged on the iBright FL1000 Imaging System.

No harmful UV rays: While UV light effectively excites many fluorescent dyes and stains, UV light is a health hazard. Furthermore, prolonged exposure to UV light can damage DNA samples, and compromise the integrity of samples to be used for downstream applications, such as subcloning.

No mercury waste: UV transilluminator bulbs contain mercury, a hazardous substance, and therefore require special care for handling and disposal.

Longer lifetime: LED bulbs have a substantially longer real-time life than UV bulbs, which can add up to considerable cost savings over the lifetime of the instrument.

Big performance in a simple package—iBright Analysis Software expands upon the on-board analysis features of the iBright Imaging Systems, with in-depth image adjustment and data analysis capabilities. iBright Analysis Software was designed to complete the overall intuitive imaging experience.

Figure 13. iBright Analysis Software features extensive analysis and image adjustment functions.

Cloud connectivity helps to increase productivity—iBright Analysis Software is built on the Thermo Fisher Cloud web-based platform, and is part of the Thermo Fisher Connect suite. Data can be exported directly from iBright Imaging Systems and securely stored in Thermo Fisher Cloud. As the iBright Analysis Software is web-based, you can access, review, analyze, and share your data wherever an internet connection is available. Furthermore, with Thermo Fisher Connect you can determine instrument status, firmware edition, and usage history, providing an extra degree of control over the monitoring of your investment.

Keep your data secured—Thermo Fisher Connect is powered by Thermo Fisher Cloud, which uses powerful security standards, including high levels of encryption and network firewalls, so your data remain secure. Thermo Fisher Cloud also provides data backup and recovery so that even in emergencies, your data remain secure. 

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