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Pack size 1 kit (for 1000 cm² membrane)
Applications Western blot


Product Name Enhanced Chemiluminescent Reagent Kit (Anti-Mouse IgG)
Synonyms ECL Anti-Mouse Antibody Detection Reagent System; ECL Western Blotting Substrate Reagent Kit (Anti-Mouse IgG); ECL-WB Substrate Reagent System (Anti-Mouse IgG); Enhanced Chemiluminescent Western Blot Related HRP-Conjugated Goat Anti-Mouse IgG Assay System
Description Enhanced Chemiluminescent Reagent Kit (Anti-Mouse IgG) is a four-component system for sensitive detection of membrane-immobilized proteins on western blots probed with mouse primary antibodies using HRP-conjugated anti-mouse secondary antibodies and enhanced chemiluminescent peroxidase substrate reagents.
Application Western blot
Pack Size 1 kit (for 1000 cm² membrane)
Assays Per Kit Sufficient for covering up to 1000 cm² of PVDF or nitrocellulose membrane
Reagent TypeProtein assay kit; ECL Western Blotting related

Kit Components*

Kit Component Component Description Size
Blocking buffer Nonfat dry milk protein powder 10 g
HRP-conjugated secondary antibody, affinity purified, solid phase adsorbed HRP-conjugated goat anti-mouse IgG 0.1ml
Enhanced chemiluminescent (ECL) reagent A HRP chemiluminescent substrate: luminol-based; enhancer 5 ml
Enhanced chemiluminescent (ECL) reagent B chemiluminescent substrate oxidizing agent 5 ml

*Additional components can be purchased. If you need extra of the above components please order them together to avoid additional shipping charges.

Assay Information

Sample Type SDS-PAGE separated-, membrane-immobilized-, primary-antibody-probed proteins from cell/tissue lysates
Assay Type Immunoassay
Assay Purpose Detect target protein; Quantify target protein level
Technique Immunodetection of target protein by HRP-conjugated secondary antibody
Detection Method Chemiluminescent
Emission 428 nm, blue glow
Equipment Needed WB instrumentation; X-ray film cassette or a charge-coupled device (CCD) imager
Assay Sensitivity Femtomole and picogram protein quantities


Form Supplied Blocking buffer: Dry powder
HRP-Antibody conjugate: Concentrated stock solution, 0.1 mg/ml, in 0.01M PBS (pH7.4) and 50% glycerol
ECL reagents A and B: Concentrated stock solutions, 20x
Recommended Working Concentration See protocol in Datasheet
Storage 4˚C for one year

Main Advantages

Specific High signal-to-noise ratio
Sensitive Up to 10 times more sensitive than colorimetric methods
Quantitative Linear response in the range 0.2 to 2 OD units
Stable The light output signal remains detectable up to several hours after incubation
Reliable Possibility for repeated exposure to X-ray film until obtain optimal results
Storable Immediate hard copy data - avoid fading of results
Reuseable Allowing reprobing on membranes up to 10 times - saves time and materials
Fast Short reaction times; few sec to few min of exposure enough to detect signal
Non-radioactive and Non-toxic No specialized facilities needed


Chemiluminescence refers to the light emitted by a chemical reaction and chemiluminescent systems have emerged as the best all-around technique for the revelation step of blotting-based analyses. Horseradish Peroxidase (HRP) Conjugated Secondary Antibodies are the most popular antibody conjugates and are widely used to obtain chemiluminescent results in western and other immunoblotting, ELISA, and immunochemistry. In the chemiluminescence reaction Horseradish peroxidase (HRP) catalyzes the oxidation of cyclic diacylhydrazides, such as luminol, or acridine derivatives in the presence of hydrogen peroxide (H2O2) acting as activator or oxidizing agent. Immediately following the oxidation, the luminol is in an excited state (intermediate reaction product), which decays to the ground state by emitting light. Strong up to 1000-fold enhancement of the light emission is produced by enhancers, such as phenolic compounds, making the light emission much prolonged and very intense. This allows detection of protein at a level of 1-10 pg.

Enhanced Chemiluminescent (ECL) Reagent Kit is an immunodetection system that utilizes horseradish peroxidase (HRP) conjugated anti-mouse, anti-rabbit, anti-goat, or anti-rat secondary antibodies for ECL western blot detection of proteins. Proteins from various samples such as cell lysates are first separated within an acrylamide gel under adapted electric current (electrophoresis) and then transferred onto a PVDF or nitrocellulose membrane (transfer). The membrane containing the proteins is then incubated with specific primary antibodies which bind to their specific target(s) (capture) and subsequently with Horseradish Peroxidase (HRP)-conjugated secondary antibodies which bind to the primary antibodies (detection). Addition of chemiluminescent reagents (HRP luminol-based substrate, enhancer and activator) results in a chemiluminescent signal that can be captured using photographic or other imaging methods (revelation). The peroxidase-catalyzed oxidation of luminol produces a glow-type light emission at 428 nm. The intensity of light output is proportional to peroxidase activity and is a measure of the number of enzyme molecules reacting and thus of the amount of target antigen.

Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG) Images

Click the images to enlarge.

Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)
Western blot - Enhanced Chemiluminescent Reagent Kit (Mouse IgG) (EK1001)

Goat Anti-Mouse IgG Secondary Antibody, HRP
at 0.2ug/ml ECL exposure

Lane 1: Mouse IgG Protein 80ng
Lane 2: Mouse IgG Protein 40ng
Lane 3: Mouse IgG Protein 20ng
Lane 4: Mouse IgG Protein 10ng
Lane 5: Mouse IgG Protein 5ng
Lane 6: Mouse IgG Protein 2.5ng
Lane 7: Mouse IgG Protein 1.25ng
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)
Enhanced Chemiluminescent (ECL) Detection Mechanism
Enhanced Chemiluminescent (ECL) Detection Kit (Mouse IgG)
Enhanced Chemiluminescent Reagent Kit (Mouse IgG) components:
Blocking buffer: 10g
HRP-conjugated goat anti-mouse IgG: 0.1 ml
Chemiluminescent reagent A: 5 ml
Chemiluminescent reagent B: 5ml
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Customer Q&As

Q: Is it possible to dilute 10X TBS stock that is 250mM Tris Base/Tris-Hcl, 27 mM KCl, and 1.37M NaCl to be used as the wash buffer?
A: You can dilute the 10X TBS stock to 1XTBS and test the pH value of the 1xTBS.
The 1XTBS can be used if the pH value falls in the range of 7.2-7.6.
Q: Would your kits work normall with samples from Wistar Rat?
A: Our kits would work normally with samples from Wistar Rat.
Q: How can I store the dissolved wash buffer for a longer period of time?
A: You can prepare a concentrated wash buffer (25X) which can be stored at 4°C for 1-3 months.
Q: Does the TMB color developing agent contain H2O2?
A: Yes, the TMB color developing agent contain H2O2.
Q: Does this ELISA kit contain any product produced in humans or primates?
Keywords: component, ingredient
A: None of the components in our ELISA kits are produced in humans or primates.
Q: What is the volume of the recombinant protein control? What is its shelf life?
Keywords: expiration, storage, temperature, size
A: The volume of the control is around 100pg. If unopened, the shelf life of the control is the same as the whole kit - "Store at 4˚C for 6 months, at -20˚C for 12 months." The reconstituted control can be stored at -20˚C for 2 days.
Q: Will it be okay to run the experiment if I accidentally used the wrong buffer (e.g. sample diluent buffer) for antibody dilution instead of the antibody diluent buffer? Keywords: dilution, replace, substitute, sample buffer
A: Using the sample diluent buffer for antibody dilution instead of the antibody diluent buffer will negatively affect the test result to some extent. The values of both standard and sample might be lower than the normal values.
Q: The protocol says to use neutral PBS and provides a recipe. Could I use neutral PBS made with KCl or KH2PO4 (common constituents for most PBS recipes) instead? Keywords: protocol, alternative buffer, applications, reagent recipe
A: Yes, it is ok to use common PBS recipes. We have tried many types of buffers with common constituents, and no significant difference was observed whatsoever.
Q: Why are my O.D. values different than your values on the datasheet? Keyword: troubleshooting, reference
A: We detected the kit in our lab and got our values on the datasheet before delivery, but protein activity will decrease as time goes on, so you may get lower O.D. values than ours. However, it should still be in a reasonable range and the standards can be used to calculate sample values. Good linearity of curve is more important than the actual numerical value.
Q: On the ELISA kit datasheet it says "HRP substrate TMB was used to visualize HRP enzymatic reaction." What is HRP and which part of the kit contains HRP? Keyword: kit components, reagents, protocol
A: It means Avidin-Biotin-Peroxidase Complex(AR1103) and you could find it in Kit Components.
Q: Do you offer any ELISA kits that can work with whole blood samples instead of plasma or urine? Keyword: applications
A: We test serum and plasma routinely, and there is very little difference between serum, plasma and whole blood. The whole blood also contains proteins we need to test. The kit can be used to test whole blood in theory.
Q: Does silicic acid (formula SiOH4) affect the results of the ELISA assay? Keyword: protocol, reagents
A: The acid may affect the binding of antigen to antibody, it is not recommended to use. That being said, it is unlikely to affect the reaction if the solution remains an overall neutral environment.
Q: Is there lot-to-lot variation of the ELISA kits? What are Boster's general services when I have questions about your kits? Keyword: technical support, help, customer service
A: The NIBSC/WHO 1st International Standard is evaluated, we always test our kits before delivery, and customers can find the test result on the protocol. If the customer needs technical support from us to analyze their data, please contact us and we ask that you provide us the following information: the lot# and production date, and when did the customer detect the kit.
Q: The results of my standards do not look correct, what could be the problem? Keyword: troubleshoot, protocol
A: Double check the protocol for plate washing. It should be examined on three aspects: 1) Did you make the washing buffer correctly? 2) How long did you let the buffer stay in wells? 3) What was the volume of the washing buffer added to each well? In addition, pay attention when adding sample to avoid contamination. And we suggest testing the standards again. Values of standards at low concentration are more affected than that at higher concentration, so customer can still get expected values at high concentration even if there is an error. If problems persist, please contact technical support with the catalog and lot number of your product.
Q: What is the well depth of the 96 well plates for the ELISA kits? Keyword: well capacity, product size
A: The well depth is 300ul, and the max capacity is 350ul. The height of the well is 1.1 cm, and the internal size is 1 cm.
Q: The kit does not include wash solution, what should I do? Keyword: wash buffer
A: Our Elisa kits do not come with wash solution, but we have included information about how to make washing buffer on the datasheet. Please refer to the "Material Required But Not Provided" section. We also offer washing buffer for sale separately (Phosphate Buffered Saline Powder SKU: AR0030).
Q: For your ELISA kits, are the capture and detection antibodies polyclonals or monoclonals? Keyword: antibody clonality
A: This information can be found for each kit under the "Properties" section, and you can find the immunogen sequence information in the "Overview" section.
Q: Do your ELISA kits come with sealants or plate covers? Keyword: storage, sequential use
A: The kit can be used within a month sequentially if it's opened and stored at 4 degree. There is no need to use sealants, the plate can be packaged with aluminum foil bag, and for other reagents keep bottle tightly closed.
Q: What is the concentration of Sodium Azide in your Elisa kits? Keyword: preservative
A: Our Elisa kits contain 0.02% Sodium Azide. All of the components except TMB colour developing reagent and stop solution contains 0.02% Sodium azide as the preservative.
Q: Are there positive and negative controls available for my ELISA kit? Keyword: positive control, negative control
A: We can provide a recombinant protein as a control. It costs $50 per control and takes 2 weeks to manufacture. We cannot provide a positive or negative control in serum.
Q: Why do I get positive results for my knockout (KO) model when used as a control?
A: The knockout (KO) model may contain truncated forms of the target protein which can be detected by the ELISA assay.
Q: How long do I soak my plate in the wash buffer?
A: The plate should soak in 0.3mL PBS or TBS wash buffer for at least 1-2 minutes in an automated wash station.
Q: Can I use Tween in my wash buffer?
A: While it is not recommended to use Tween in your wash buffer, small amounts (<0.1% concentration) may decrease background due to insufficient blocking.
Q: What plate type do I use to set up the microplate reader?
A: Our plates are made with the Corning costar plates similar to the DNA-BIND 96 -well plates.
Q: What should I use for negative control?
A: Please contact us for negative control suggestions. You can also check expression databases such as genecards, uniprot etc. Due to logistic reasons, we do not sell serum or lysates that we use internally for positive or negative control.
Q: Where can I find troubleshooting information? What should I do if I have unexpected bands, high background, no signal, weak signal
A: You can find Boster's troubleshoot guides under tech support tab. Please contact us for further assistance on troubleshooting your experiment.
Q: What is the normal level of this protein in my sample of interest?
A: We have reviewed literature and have gathered this information for most of our ELISA kits. You can find this information on the product page or contact us if you cannot find it. However this information is only suggestive and cannot replace pilot studies in determining the optimal sample dilution ratio.
Q: Is the plate separable? Can I use only part of the kit and save the rest for later? How many samples can I run with one kit?
A: Yes the plate is separable. There are 12 strips of 8 wells each, all removable from the plate. The amount of samples you can run depend on a few factors. In the most common ELISA set up, you will use two strips for standards, and 10 strips for samples using duplicates, which let you run up to 40 samples per kit. Contact us if you have questions regarding other situations.
Q: Does the kit contain sample preparation reagents? How do I prepare my samples?
A: Since different sample types require different reagents, we do not include them in the ELISA kit. However we do have each reagent mentioned in the file below available at very reasonable prices. Be sure to check them out. For sample preparation protocols please download the file below:
Q: Can this ELISA kit work on brain tissue homogenate, cell culture supernatant, saliva, urine, serum, whole blood or any other sample type?
A: In theory the ELISA kit will work for all sample types if the target protein is present at a level that falls within the linear range of the ELISA kit detection range. We guarantee the kit to work on the sample types that we have tested. If you want dilution ratio suggestions on these sample types please contact our technical support. For sample types that we have not tested for, we suggest you run pilot experiments to decide the optimal sample dilution.
Q: Can this ELISA kit react with the pro-form of the target protein? Can this ELISA kit react with an isoform of the protein?
A: In general, unless otherwise specified, the ELISA kit is pan-specific, meaning that it will react with all different forms of the target protein if they share the majority of the target protein's sequence. The capture and detection antibodies are reactive to the entire sequence of the standard protein. You can find the sequence information of the standard protein in the "immunogen" section. For more information about the specificity of the kit for your particular experiment, please contact our techincal support.
Q: Can this ELISA kit react with human, mouse, rat or other species?
A: If the kit is reactive to another commonly used species (human, mouse, and/or rat), we would have listed it as a separate product. If you want to check cross-reactivity to a species that is not included in the 3 species listed above, please contact our technical support for more information. As a rule of thumb, if the sequences are 90%+ identical, there is a high chance of cross-reactivity for your species of interest.