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Detection
Fluorescent western blots
Chemiluminescent
western blots
Colorimentric western blots
The last step in the western blotting workflow (after the separation of proteins by PAGE and their transfer from gel to membrane) is detection.
In this step, primary antibodies specific to the protein of interest bind to the protein on the membrane. With a variety of detection techniques to choose from (including chemiluminescence, fluorescence, or chromogenic detection), performers of western blot analysis can select a technology that matches experimental requirements and available instruments. Quick visualisation or precise quantitation, single-probe detection or multiplexing—we offer a range of reagents and kits for western blot detection and subsequent analysis.
Buffers and blotting reagents
Thermo Scientific StartingBlock Blocking Buffer* |
Thermo Scientific StartingBlock Blocking Buffer* |
Thermo Scientific Blocker FL Fluorescent Blocking Buffer |
Thermo Scientific SEA BLOCK Blocking Buffer |
Thermo Scientific Pierce Protein-Free Blocking Buffer* |
| New target | Biotin-aided detection | Recommended for fluorescence | Specialty | |
Broad compatibility Compatible with most antibodies as well as biotin-binding reagent |
Fast Block membranes in 5–10 minutes |
Designed to reduce cross-reactivity and produce high signal-to-noise ratios |
Fish serum, less likely to have nonspecific binding with antibodies and other mammalian proteins |
Proprietary nonprotein blocking compound— eliminate potential cross-reactivity associated with protein-based blockers and provide high signal-to-noise ratios |
High signal-to-noise ratio biotin- and serum-free formulation, can achieve signal-tonoise ratios from 10:1 to 20:1 |
Low background The nonserum protein solution yields a high signal-to-noise ratio |
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Invitrogen products
Thermo Scientific Surfact-Amps detergents
Restore Stripping Buffer |
Restore PLUS Stripping Buffer |
Restore Fluorescent Western Blot Stripping Buffer |
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| Features | Gentle, odor-free | Robust yet gentle, odor-free |
Optimized for near-infrared (NIR) fluorescent blotting |
| Membrane | NC and PVDF | NC and PVDF | Use with low-fluorescence PVDF membrane, Cat. No. 22860 |
| Incubation Time | 15–30 minutes at room temperature |
5–15 minutes at room temperature |
5–15 minutes at room temperature |
| Select when | Primary antibody is susceptible to stripping buffers |
Using high-affinity primary antibody |
Using NIR-labeled antibody |
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- Membrane compatibility
Compatible with PVDF and nitrocellulose membranes - Substrate compatibility
Verified for use with chromogenic, chemiluminescent, and fluorescent detection methods
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Invitrogen products
Invitrogen products
Substrate reagents
Among the six types of chemiluminescent substrates for western blot detection with HRP, we have five majors:
For data above: STAT3 detection in HeLa cell lysate (lane 1: 20 μg total protein; lanes 2–6: serially diluted 1:1) was performed using Thermo Scientific™ HRP chemiluminescent substrates. The blots were developed using Invitrogen™ Anti-STAT3 Antibody (Cat. No. MA1-13042) and Goat Anti-Mouse IgG Secondary Antibody, HRP conjugate (Cat. No. 31430). Images were captured using a CCD camera-based imaging instrument.
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- Picogram-to-femtogram sensitivity
Detect low-picogram to high-femtogram amounts of target protein on nitrocellulose or PVDF membrane - Enhanced stability
Stable signal duration over the critical 4-hour time period with signal light output up to 24 hours - Exceptional robustness
High performance, even outside of the recommended antibody ranges, including the most commonly used 1:5,000 to 1:10,000 secondary antibody dilutions from a 1 mg/mL stock solution - Sensitivity
The SuperSignal West Pico PLUS substrate has better sensitivity than Bio-Rad Clarity substrate (see below)
- SuperSignal West Pico PLUS substrate outperforms Bio-Rad Clarity substrate
Detection of the indicated targets was performed using two-fold serial dilutions of HEK 293 or HeLa cell lysates, starting with the amount indicated in parentheses. Following separation by SDS-PAGE, proteins were transferred to either Thermo Scientific PVDF (Cat. No. 88518) or nitrocellulose (Cat. No. 88018) membranes using the Invitrogen Power Blotter XL (Cat. No. PB0013) and Thermo Scientific Pierce 1-Step Transfer Buffer (Cat. No. 84731). The membranes were blocked with 5% nonfat dry milk dissolved in Thermo Scientific Pierce 20X TBS Tween 20 Buffer (Cat. No. 28360), and incubated with Invitrogen antibodies against beta-Catenin (Cat. No. MA1-300; 1:5,000), eIF4E (Cat. No. MA1-089; 1:1,000), RSK2 (Cat. No. MA5-15920; 1:1,000), or Ezrin (Cat. No. MA5-13862; 1:1,000), followed by incubation with Invitrogen Goat Anti-Mouse IgG (H+L) Secondary Antibody, HRP Conjugate (Cat. No. 31430) at a concentration of 20 ng/mL. Chemiluminescent detection and substrate comparison was performed following a 5-minute incubation with either SuperSignal West Pico PLUS or Bio-Rad Clarity substrate. Signal was captured using X-ray film
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- 24-hour light emission—
10 times longer than with other enhanced chemiluminescent substrates for HRP; make multiple exposures for publicationquality blote - Great sensitivity
With femtogram-level detection, see bands you’ve never been able to see before - Save your antibody
Requires very dilute antibody concentrations, allowing you to perform 25-50 times more blots than with other chemiluminescent substrates - Intense
Immediate stable signal generation provides easy detection on film or compatible imaging system - Working solution is stable for at least 24 hours
The kit itself is stable for at least one year
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- Low-femtogram sensitivity
Low-femtogram to high-attogram ultrasensitivity - Excellent signal duration
Up to 6 hours of usable light output when conditions are optimised - Superior sensitivity and signal-to-noise ratio
- Exhibits superior intensity and sensitivity to detect proteins that were previously undetectable using other high-performance ECL substrates on the market
- SuperSignal West Atto shows superior sensitivity compare to Clarity Max substrates providing a better signal-to-noise ratio
- The SuperSignal West Atto Ultimate Sensitivity Substrate offers longer signal duration (6 hours vs. 2), which allows for the acquisition of multiple exposures to more easily obtain publication-quality blot images
- Sensitivity
The SuperSignal West Atto Ultimate Sensitivity substrate has better sensitivity than Bio-Rad Clarity Max substrate and Millipore Immobilon (see below)
Figure 1. SuperSignal West Atto substrate enables detection of even low concentrations of target.
Serial dilutions of HeLa lysate were prepared, separated by SDS-PAGE, and transferred to nitrocellulose membranes for detection of p23. The protein was detected using an Invitrogen mouse anti-p23 primary antibody (Cat. No. MA3-414) followed by an Invitrogen HRP-conjugated goat anti-mouse secondary antibody (Cat. No. 32430) diluted per manufacturer’s instructions. Blots were then incubated with SuperSignal West Atto substrate (Cat. No. A38558), Clarity Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.), or Immobilon Western Chemiluminescent HRP Substrate (MilliporeSigma) per product instructions. All blots were simultaneously imaged using the Invitrogen iBright FL Imaging System (A). Compared to the amounts of lysate required for detectable signal using the other substrates, only 1/16 of the amounts is needed for comparable signal intensities using the SuperSignal West Atto substrate (B), conserving precious samples.
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Antibodies
- Superior sensitivity and low background for improved detection of low-abundance targets
- Improved dye chemistry combined with cross-adsorption for enhanced multiplexing capabilities
- Ready to use—no need to reconstitute
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Alexa Fluor Plus secondary antibodies
Goat anti-Rabbit
Goat anti-Mouse
Goat anti-Chicken
Donkey anti-Goat
Donkey anti-Rabbit
Donkey anti-Mouse
X-ray films
- High performance
Just as sensitive as other commercially available autoradiography films - Excellent signal duration
Costs up to 75% less than other brands of comparable-quality X-ray film - Convenient
Available in five sheet sizes in packages containing 50 or 100 non-interleaved sheets
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For Research Use Only. Not for use in diagnostic procedures.