As with other components in a western blotting system, there are many chemiluminescent western blotting substrate choices available. The appropriate selection of a substrate depends on the detection level (sensitivity) required, the target protein's abundance, the sample abundance and antibody availability. Use the tables below to find the right horseradish peroxidase (HRP) or alkaline phosphatase (AP) substrate for your experimental needs.
Recommended Chemiluminescent Substrates
 |  |  | |
| SuperSignal West Atto | SuperSignal West Pico PLUS | SuperSignal West Dura | |
|---|---|---|---|
| Choose when… | Target is very low-abundant, sample is limited, or antibodies are limited | Everyday applications; offers improved sensitivity over base level ECL | Performing quantitative western blotting or maximum signal duration is needed |
| Features | Highest sensitivity | Widest dynamic range | Longest duration and linearity |
| Sensitivity | Low femtogram to high attogram | Low picogram to femtogram | Mid femtogram |
| Duration | 6 hours | up to 24 hours | 24 hours |
| Recommend antibody dilutions (1 mg/ml stock) | 1°: 1:5,000 2°: 1:100,000-1:250,000 | 1°: 1:1,000 2°: 1:20,000-1:100,000 | 1°: 1:5,000 2°: 1:50,000-1:250,000 |
| Cat. No. | A38554 (20 mL) A38555 (100 mL) A38556 (200 mL) | 34579 (20 mL) 34577 (200 mL) 34580 (500 mL) 34578 (1 L) | 37071 (20 mL) 34075 (100 mL) 34076 (200 mL) |
| Explore application data | Explore application data | Explore application data |
Additional products for HRP substrates
Horseradish peroxidase (HRP) chemiluminescent substrates
HRP (40 kDa) has become the standard enzyme for chemiluminescent western blot detection. Its popularity grew due to its stability and smaller size, which enables more molecules conjugated per IgG providing greater sensitivity. Furthermore, advancement and improvements in chemiluminescent substrates for HRP has enabled even higher sensitivity over AP substrates for western blotting. Choice of the HRP chemiluminescent substrate used should be based on abundance of your target protein of interest, abundance of sample containing the target protein, quality of the antibodies and the level of sensitivity and type of instrumentation available for detection.
Webinar: Detecting low abundant proteins in a western blot
In this webinar, we discuss specific methods from sample preparation through immunodetection that can help overcome challenges and improve signal to noise of low-abundance proteins for more successful western blot detection.
AP substrates
Novex AP Chemiluminescent Substrate
| Long signal duration | |
|---|---|
| Sensitivity | Low picogram |
| Duration | Over 24 hours |
| Recommend antibody dilutions (1 mg/ml stock) | 1°: 1:500-5,000 2°: 1:2,000-1:10,000 |
| When to use | Used standalone substrate for PVDF membranes Add Nitro Block II (sold separately) to substrate for use with nitrocellulose membranes |
Additional products for AP substrates
| Thermo Fisher Scientific product | Description | Cat. No. |
|---|---|---|
| Novex AP Chemiluminescent Substrate Enhancer (Nitro Block II) | 20X solution of Nitro-Block-II intended for use in conjunction with Novex AP Chemiluminescent Substrate to increase the intensity of chemiluminescent signals when probing blots on nitrocellulose membranes | WP20003 |
| WesternBreeze Chromogenic Kit, anti-rabbit | Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using mouse primary antibodies | WB7104 |
| WesternBreezeChemiluminescent Kit, anti-rabbit | Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using rabbit primary antibodies | WB7106 |
| WesternBreeze Chemiluminescent Kit, anti-goat | Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using goat primary antibodies | WB7108 |
Alkaline phosphatase (AP) chemiluminescent substrates
For western blot detection based on alkaline phosphatase (AP), 86 kDa, we offer our CDP-Star substrate that is designed to deliver picogram level sensitivity and is compatible with both traditional x-ray film and CCD-based imaging. CDP-Star is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases. The emitted light is stable up to 24-96 hours allowing for multiple exposures.
Choose from a standalone substrate, Novex AP Chemiluminescent Substrate or the WesternBreeze Chemiluminescent Immunodetection kits which contains all solutions necessary for your application including blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution, ready-to-use chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter papers, polyester sheet for even substrate development on the membrane. Novex AP Chemiluminescent Substrate should be combined with Nitro Block II for use with nitrocellulose membranes.
Figure 1. Light Emission mechanism for CDP-Star Substrate.
Nitrocellulose membranes provide an inefficient environment for chemiluminescence from 1,2-dioxetane substrates, resulting in very low signal intensity. Nitro-Block-II membrane enhancer increases signal intensity on both nitrocellulose and PVDF membranes. Nitro-Block-II enhancers generate a hydrophobic environment on the membrane surface that increases the intensity of chemiluminescence. The effects of NitroBlock enhancer treatment on nitrocellulose and PVDF membranes are shown. Without Nitro-Block enhancer, the signal on nitrocellulose membranes is weak, making detection either impossible or requiring extremely long exposure times. With Nitro-Block enhancer, short exposures of 10 to 45 minutes are achieved. While PVDF membranes does not require the use of Nitro-Block enhancer, exposure times are tenfold faster with Nitro-Block enhancer. This permits very short exposure times on PVDF, ranging from 15 seconds to 15 minutes for Western blots.
Figure 2. Chemiluminescent signal enhancement on Nitrocellulose and PVDF membranes with Nitro-Block Enhancer using a short exposure time.
Product manuals
Application notes and brochures
Get started with our combo kits
SuperSignal West Substrate Combo Kit
| Option A |
|---|
Pico PLUS (500mL) + Trial size Atto (20mL) This combo kit includes:
|
| Option B |
|---|
Femto (100 mL) + Trial size Atto (20 mL) This combo kit includes:
|
| Option C |
|---|
Atto (100 mL) + Trial size Pico PLUS (20 m) This combo kit includes:
|
| Option D |
|---|
Pico PLUS (20 mL) and Atto (20 mL) This combo kit includes:
|
Image capture and analysis for chemiluminescent western blots
Chemiluminescent western blot signal can be captured with X-ray film, charge-coupled device (CCD) camera–based digital imaging instruments, and phosphorimagers that detect chemiluminescence. X-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, CCD camera–based imaging instruments offer the advantages of qualitative analysis, instant image capture and analysis, higher sensitivity, greater resolution and a larger dynamic range than film.
iBright Imaging Systems
Capture and analyze publication quality images from chemiluminescent western blots with iBright Imaging Systems. These high-performance instruments enhance the imaging experience through powerful hardware, advanced automated technologies, and an interface that is easy to use for researchers of all experience levels.
X-Ray film
Thermo Scientific CL-XPosure Film is an excellent photographic film for use with enhanced chemiluminescence (ECL) substrates for horseradish peroxidase (HRP) or alkaline phosphatase (AP).
Recommended Chemiluminescent Substrates
| | | |
| SuperSignal West Atto | SuperSignal West Pico PLUS | SuperSignal West Dura | |
|---|---|---|---|
| Choose when… | Target is very low-abundant, sample is limited, or antibodies are limited | Everyday applications; offers improved sensitivity over base level ECL | Performing quantitative western blotting or maximum signal duration is needed |
| Features | Highest sensitivity | Widest dynamic range | Longest duration and linearity |
| Sensitivity | Low femtogram to high attogram | Low picogram to femtogram | Mid femtogram |
| Duration | 6 hours | up to 24 hours | 24 hours |
| Recommend antibody dilutions (1 mg/ml stock) | 1°: 1:5,000 2°: 1:100,000-1:250,000 | 1°: 1:1,000 2°: 1:20,000-1:100,000 | 1°: 1:5,000 2°: 1:50,000-1:250,000 |
| Cat. No. | A38554 (20 mL) A38555 (100 mL) A38556 (200 mL) | 34579 (20 mL) 34577 (200 mL) 34580 (500 mL) 34578 (1 L) | 37071 (20 mL) 34075 (100 mL) 34076 (200 mL) |
| Explore application data | Explore application data | Explore application data |
Additional products for HRP substrates
Horseradish peroxidase (HRP) chemiluminescent substrates
HRP (40 kDa) has become the standard enzyme for chemiluminescent western blot detection. Its popularity grew due to its stability and smaller size, which enables more molecules conjugated per IgG providing greater sensitivity. Furthermore, advancement and improvements in chemiluminescent substrates for HRP has enabled even higher sensitivity over AP substrates for western blotting. Choice of the HRP chemiluminescent substrate used should be based on abundance of your target protein of interest, abundance of sample containing the target protein, quality of the antibodies and the level of sensitivity and type of instrumentation available for detection.
Webinar: Detecting low abundant proteins in a western blot
In this webinar, we discuss specific methods from sample preparation through immunodetection that can help overcome challenges and improve signal to noise of low-abundance proteins for more successful western blot detection.
AP substrates
Novex AP Chemiluminescent Substrate
| Long signal duration | |
|---|---|
| Sensitivity | Low picogram |
| Duration | Over 24 hours |
| Recommend antibody dilutions (1 mg/ml stock) | 1°: 1:500-5,000 2°: 1:2,000-1:10,000 |
| When to use | Used standalone substrate for PVDF membranes Add Nitro Block II (sold separately) to substrate for use with nitrocellulose membranes |
Additional products for AP substrates
| Thermo Fisher Scientific product | Description | Cat. No. |
|---|---|---|
| Novex AP Chemiluminescent Substrate Enhancer (Nitro Block II) | 20X solution of Nitro-Block-II intended for use in conjunction with Novex AP Chemiluminescent Substrate to increase the intensity of chemiluminescent signals when probing blots on nitrocellulose membranes | WP20003 |
| WesternBreeze Chromogenic Kit, anti-rabbit | Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using mouse primary antibodies | WB7104 |
| WesternBreezeChemiluminescent Kit, anti-rabbit | Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using rabbit primary antibodies | WB7106 |
| WesternBreeze Chemiluminescent Kit, anti-goat | Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using goat primary antibodies | WB7108 |
Alkaline phosphatase (AP) chemiluminescent substrates
For western blot detection based on alkaline phosphatase (AP), 86 kDa, we offer our CDP-Star substrate that is designed to deliver picogram level sensitivity and is compatible with both traditional x-ray film and CCD-based imaging. CDP-Star is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases. The emitted light is stable up to 24-96 hours allowing for multiple exposures.
Choose from a standalone substrate, Novex AP Chemiluminescent Substrate or the WesternBreeze Chemiluminescent Immunodetection kits which contains all solutions necessary for your application including blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution, ready-to-use chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter papers, polyester sheet for even substrate development on the membrane. Novex AP Chemiluminescent Substrate should be combined with Nitro Block II for use with nitrocellulose membranes.
Figure 1. Light Emission mechanism for CDP-Star Substrate.
Nitrocellulose membranes provide an inefficient environment for chemiluminescence from 1,2-dioxetane substrates, resulting in very low signal intensity. Nitro-Block-II membrane enhancer increases signal intensity on both nitrocellulose and PVDF membranes. Nitro-Block-II enhancers generate a hydrophobic environment on the membrane surface that increases the intensity of chemiluminescence. The effects of NitroBlock enhancer treatment on nitrocellulose and PVDF membranes are shown. Without Nitro-Block enhancer, the signal on nitrocellulose membranes is weak, making detection either impossible or requiring extremely long exposure times. With Nitro-Block enhancer, short exposures of 10 to 45 minutes are achieved. While PVDF membranes does not require the use of Nitro-Block enhancer, exposure times are tenfold faster with Nitro-Block enhancer. This permits very short exposure times on PVDF, ranging from 15 seconds to 15 minutes for Western blots.
Figure 2. Chemiluminescent signal enhancement on Nitrocellulose and PVDF membranes with Nitro-Block Enhancer using a short exposure time.
Product manuals
Application notes and brochures
Get started with our combo kits
SuperSignal West Substrate Combo Kit
| Option A |
|---|
Pico PLUS (500mL) + Trial size Atto (20mL) This combo kit includes:
|
| Option B |
|---|
Femto (100 mL) + Trial size Atto (20 mL) This combo kit includes:
|
| Option C |
|---|
Atto (100 mL) + Trial size Pico PLUS (20 m) This combo kit includes:
|
| Option D |
|---|
Pico PLUS (20 mL) and Atto (20 mL) This combo kit includes:
|
Image capture and analysis for chemiluminescent western blots
Chemiluminescent western blot signal can be captured with X-ray film, charge-coupled device (CCD) camera–based digital imaging instruments, and phosphorimagers that detect chemiluminescence. X-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, CCD camera–based imaging instruments offer the advantages of qualitative analysis, instant image capture and analysis, higher sensitivity, greater resolution and a larger dynamic range than film.
iBright Imaging Systems
Capture and analyze publication quality images from chemiluminescent western blots with iBright Imaging Systems. These high-performance instruments enhance the imaging experience through powerful hardware, advanced automated technologies, and an interface that is easy to use for researchers of all experience levels.
X-Ray film
Thermo Scientific CL-XPosure Film is an excellent photographic film for use with enhanced chemiluminescence (ECL) substrates for horseradish peroxidase (HRP) or alkaline phosphatase (AP).
Chemiluminescent detection
Chemiluminescent detection occurs when energy from a chemical reaction is released in the form of light. The two most common enzyme reporters that catalyze chemiluminescent reactions required for generating a recordable signal are horseradish peroxidase (HRP) and alkaline phosphatase (AP).
| HRP substrates (Horseradish peroxidase) | AP substrates (Alkaline phosphatase) | |
|---|---|---|
| Sensitivity |
|
|
| Signal generation | Immediate | Gradually increases with signal maximum at ~30-60 minutes |
| Signal duration | Up to 24 h | 24-96 hours |
| Considerations | Compatible with common buffers such as TBS and PBS | Not compatible with phosphate buffers |
| When to use | Antibodies or Probes conjugated to HRP | Antibodies or Probes conjugated to AP |
Find HRP linked secondary antibodies for chemiluminescent western blotting detection ›
Find AP conjugated secondary antibodies for chemiluminescent western blotting detection ›
Resources
Download: Western blotting overview brochure
Explore: Western blot protein ladders
Explore: Western blot imaging and analysis
For Research Use Only. Not for use in diagnostic procedures.