Show Order Info
SKU AR1170
Pack size 200mL
Applications To detect antigen at low picogram level by reacting with horseradish peroxidase

List of Components

DescriptionQuantityVolumeCatalog Number
Reagent A: Luminol and luminous enhancer1100mL AR1170-A
Reagent B: Peroxide and stabilizer1100mLAR1170-B

Overview

Product Name ECL Western Blotting Substrate
SKU/Catalog Number AR1170
Form Supplied Reagent A and B: ready-to-use 1X solutions
Pack Size 200mL((sufficient reagents for 2000 cm2 of membrane)
Storage Upon receipt store ECL Western Blotting Substrate at 4°C. Protect from light. It is stable at 4°C for one year.
Detection Method Chemiluminescent
Substrate Type HRP (Horseradish Peroxidase) Substrate
Description Boster’s ECL Western Blotting Substrate is a luminol-based chemiluminescent substrate and it can detect horseradish peroxidase (HRP) at high sensitivity levels (low picogram).
Equivalent Thermofisher (Product No. 32106, 32109, 32209);
Bio-Rad Immu-StarTM HRP Chemiluminescent Kit (Product No. 170-5040, 170-5041, 170-5042, 170-5043, 170-5044, 170-5045, 170-5046, 170-5047)
Cite This Product ECL Western Blotting Substrate (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR1170)
Application To detect antigen at low picogram level by reacting with horseradish peroxidase
*Our Boster Guarantee covers the use of this product in the above tested applications.

Notes:

Type of ECL Western Blotting SubstrateSensitivity LevelCatalog Number
ECL Western Blotting Substratelow picogramAR1170
ECL Plus Western Blotting Substratelow-picogram to mid-femtogram levelAR1196

Assay Principle

The Boster Western Blotting Substrate is a luminol-based chemiluminescent substrate and it can detect horseradish peroxidase (HRP) at high sensitivity levels (low picogram). Boster Western Blotting Substrate may be used for immunoblots, western blots, dot blots and any blotting application utilizing horseradish peroxidase (HRP)-conjugates. The substrate can be used with various blocking buffers and on nitrocellulose or PVDF membranes. Such blots will exhibit low backgrounds. Produced chemiluminescence can be visualized on CCD imaging systems or x-ray film.

Additional Materials Required

1. Nitrocellulose or PVDF membranes

2. X-ray film or an imaging system

3. Rotary platform shaker for agitation of membrane during incubations

4. Wash Buffer: PBS or TBS containing 0.05-0.1% Tween-20 (PBS: 10mM Na3PO4, 150mM NaCl, pH7.2; TBS: 10mM Tris, 150mM NaCl, pH7.4)

5. Blocking Buffer: Add some blocking medium (e.g. casein, BSA or non-fat milk) into the Wash Buffer for a final blocking medium concentration of 1-5% (w/v)

6. Primary Antibody: Choose an antibody specific to the target proteins. Prepare the antibody stock solution in Wash Buffer or Blocking Buffer

7. HRP-conjugated Secondary Antibody: Choose a HRP-conjugated Secondary Antibody that specifically binds to the primary antibody.

Important Product Information

• For optimal results, use a shaking platform during incubation steps.

• Do not use sodium azide as a preservative for buffers. Sodium azide is an inhibitor of HRP.

• Do not handle the membrane with bare hands. Always wear gloves or use clean forceps.

• All equipment must be clean and free of foreign material. Metallic devices must have no visible signs of rust. Rust may cause speckling and high background.

• Exposure to the sun or any other intense light can harm the substrate. For best results keep the substrate working solution in an amber bottle and avoid prolonged exposure to any intense light. Short-term exposure to typical laboratory lighting will not harm the working solution.

• Empirical testing is essential to determine the appropriate blocking reagent for each Western blot system, as crossreactivity of the blocking reagent with antibodies can cause nonspecific signal and varying system sensitivity.

• When using avidin/biotin systems, avoid using milk as a blocking reagent as milk contains variable amounts of endogenous biotin, which causes high background signal.

• Use sufficient volumes of wash buffer, blocking buffer, antibody solution and substrate working solution to cover the blot and ensure that it never becomes dry. Using large blocking and wash buffer volumes minimizes nonspecific signal.

• Add Tween™ 20 Detergent (final concentration of 0.05-0.1%) to the blocking buffer and all diluted antibody solutions to minimize nonspecific signal.

• Do not use polystyrene vessels to mix and prepare the substrate working solution; this type of plastic causes the solution to become cloudy and produce a precipitate.

• Use new pipette tips separately while pipetting reagent A and B to avoid cross contamination.

Assay Protocol

1. Remove blot from the transfer apparatus and wash membrane with Wash Buffer 3 times for 5 minutes each.

2. Block nonspecific sites with Blocking Buffer for 60 minutes at room temperature with shaking.

3. Remove the blocking buffer and add the primary antibody working solution. Incubate blot for 1 hour at room temperature with shaking or overnight at 2-8°C with shaking.

4. Suspend membrane in Wash buffer and agitate for more than 5 minutes. Replace Wash Buffer at least 4-6 times. Increasing the volume of Wash Buffer, the numbers of washes and wash duration may help minimize background signal.

5. Incubate blot with the HRP conjugated secondary antibody working solution for 1 hour at room temperature with shaking.

6. Repeat Step 3 to remove nonbound HRP conjugate.
Note: The membrane must be thoroughly washed after incubation with the HRP conjugate.

7. Prepare the substrate working solution by mixing Reagent A and Reagent B at 1:1. Use 0.1mL working solution per cm2 of membrane.

8. Incubate blot with working solution for 1-5 minutes at room temperature.

9. Place blot in a clear plastic wrap or transparent plastic sheet protector. Use an absorbent tissue to remove excess liquid and carefully press out any bubbles from between the blot and the membrane protector.

10. Place the protected membrane in a film cassette with the protein side facing up.

11. Place X-ray film on top of the blot membrane. Perform a exposure of 1 minute, Vary the exposure time to achieve optimal results. Light emission is most intense during the first 5-30 minutes after substrate incubation.
Note: CCD detection: Put the blot membrane in the CCD and detect the chemiluminescence image according to the manufacturers’ instructions.

12. Develop and fix the film.

Troubleshooting

ProblemPossible CauseSolution
Blot glows in the dark room
Membrane has brown or yellow bands
Signal fades quickly
Too much HRP in the sysem Dilute HRP conjugate further
Weak or no signal Used insufficient quantities of antigen or antibodies
Insufficient protein transfer
Low HRP or substrate activity
Increase the amount of antigen or antibodies
Optimize transfer condition
Replace the secondary antibody or substrate
High Background Too much HRP in the sysem
Inadequate blocking or used inappropriate blocking buffer
Insufficient washing
Use too much antigen or antibodies
Use non- specific primary antibody
Overexposed film
Dilute HRP-conjugate further
Optimize blocking conditions
Increase duration, number and volume of washes
Decrease the amount of antigen or antibodies
Replace the antibody
Decrease exposure time
Spots within the protein bands Ineffective protein transfer
Unevenly hydrated membrane
Bubble between X-ray film and membrane
Optimize transfer procedure
Replace the membrane
Remove all bubbles before exposing blot to film
Speckled background Inadequate blocking Optimize blocking conditions
Nonspecific bands Too much HRP in the system
SDS caused nonspecific binding to protein
Use non-specific primary antibody
Inadequate blocking
Dilute HRP conjugate further
Do not use SDS
Replace the antibody
Optimize blocking conditions

ECL Western Blotting Substrate Images

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ECL Western Blotting Substrate
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Publications

Sequencing and Expression Characterization of Antifreeze Protein Maxi-Like in Apis cerana cerana
The Optimization of Soluble PTEN Expression in Escherichia coli
Expressional and functional interactions of two Apis cerana cerana olfactory receptors
MicroRNA-27a protects retinal pigment epithelial cells under high glucose conditions by targeting TLR4
Characterization of an Atypical Metalloproteinase Inhibitors Like Protein (Sbp8-1) From Scallop Byssus
MicroRNA-206 regulates the epithelial-mesenchymal transition and inhibits the invasion and metastasis of prostate cancer cells by targeting Annexin A2
5-Hydroxy-4?-Nitro-7-Propionyloxy-Genistein Inhibited Invasion and Metastasis via Inactivating Wnt/?-Catenin Signal Pathway in Human Endometrial Carcinoma Ji Endometrial Cells
Di-2-pyridylhydrazone Dithiocarbamate Butyric Acid Ester Exerted Its Proliferative Inhibition against Gastric Cell via ROS-Mediated Apoptosis and Autophagy
Danhong Injection Combined With t-PA Improves Thrombolytic Therapy in Focal Embolic Stroke
HSPA12B overexpression induces cisplatin resistance in non-small-cell lung cancer by regulating the PI3K/Akt/NF-?B signaling pathway
Effects of 5-hydroxy-4?-nitro-7-propionyloxy-genistein on inhibiting proliferation and invasion via activating reactive oxygen species in human ovarian cancer A2780/DDP cells
Preliminary Study of the Role F-Box Protein 32 (FBXO32) in Colorectal Neoplasms Through the Transforming Growth Factor beta (TGF-?)/Smad4 Signalling Pathway
Icariin Regulates the Bidirectional Differentiation of Bone Marrow Mesenchymal Stem Cells through Canonical Wnt Signaling Pathway
Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1?-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways
GOLPH3: a novel biomarker that correlates with poor survival and resistance to chemotherapy in breast cancer
Fibroblast growth factor 21 delayed endothelial replicative senescence and protected cells from H2O2-induced premature senescence through SIRT1
Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy
Meclizine Prevents Ovariectomy-Induced Bone Loss and Inhibits Osteoclastogenesis Partially by Upregulating PXR
Hydrogen sulfide alleviates myocardial fibrosis in mice with alcoholic cardiomyopathy by downregulating autophagy
Iguratimod prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator-activated receptor-?
Effects of celecoxib on cell apoptosis and Fas, FasL and Bcl-2 expression in a BGC-823 human gastric cancer cell line
Protective effects of heme oxygenase-1-transduced bone marrow-derived mesenchymal stem cells on reduced-size liver transplantation: Role of autophagy regulated by the ERK/mTOR signaling pathway
MicroRNA-144-3p suppresses tumor growth and angiogenesis by targeting SGK3 in hepatocellular carcinoma
The protective effects of Resveratrol against radiation-induced intestinal injury
Heme Oxygenase-1, a Key Enzyme for the Cytoprotective Actions of Halophenols by Upregulating Nrf2 Expression via Activating Erk1/2 and PI3K/Akt in EA.hy926 Cells
The hemagglutinin-neuramidinase protein of Newcastle disease virus upregulates expression of the TRAIL gene in murine natural killer cells through the activation of Syk and NF-?B
HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1-pAFP-TK and targeted killing effect
Adiponectin attenuates high glucose-induced apoptosis through the AMPK/p38 MAPK signaling pathway in NRK-52E cells
Retinoid acid-induced microRNA-31-5p suppresses myogenic proliferation and differentiation by targeting CamkII?
Fermented Chinese formula Shuan-Tong-Ling attenuates ischemic stroke by inhibiting inflammation and apoptosis
AhR-E2F1-KGFR signaling is involved in KGF-induced intestinal epithelial cell proliferation
miR-1181 inhibits invasion and proliferation via STAT3 in pancreatic cancer
Inhibition of Autophagy by Deguelin Sensitizes Pancreatic Cancer Cells to Doxorubicin
Fermented Chinese Formula Shuan-Tong-Ling Protects Brain Microvascular Endothelial Cells against Oxidative Stress Injury
Epoxyeicosanoids prevent intervertebral disc degeneration?in vitro?and?in vivo
miR-138-5p suppresses autophagy in pancreatic cancer by targeting SIRT1
Smad3-related miRNAs regulated oncogenic TRIB2 promoter activity to effectively suppress lung adenocarcinoma growth
Disrupted in schizophrenia 1 (DISC1) inhibits glioblastoma development by regulating mitochondria dynamics
miR-20b Inhibits T Cell Proliferation and Activation via NFAT Signaling Pathway in Thymoma-Associated Myasthenia Gravis
Monoacylglycerol Lipase: A Novel Potential Therapeutic Target and Prognostic Indicator for Hepatocellular Carcinoma
Downregulation of ZEB2-AS1 decreased tumor growth and metastasis in hepatocellular carcinoma
CXCL12/CXCR4 Axis Regulates Aggrecanase Activation and Cartilage Degradation in a Post-Traumatic Osteoarthritis Rat Model
Ocular Albinism Type 1 Regulates Melanogenesis in Mouse Melanocytes
Hepcidin in non-alcoholic fatty liver disease regulated by the TLR4/NF-?B signaling pathway
siRNA targeting RBP2 inhibits expression, proliferation, tumorigenicity and invasion in thyroid carcinoma cells
Amlexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss
Electromagnetic radiation at 900?MHz induces sperm apoptosis through bcl-2, bax and caspase-3 signaling pathways in rats
RUNX2 RNA interference inhibits the invasion of osteosarcoma
3,4-dihydroxyphenylethanol suppresses irradiation-induced pulmonary fibrosis in adult rats
ROS/Autophagy/Nrf2 Pathway Mediated Low-Dose Radiation Induced Radio-Resistance in Human Lung Adenocarcinoma A549 Cell
MicroRNA-27a-3p Inhibits Melanogenesis in Mouse Skin Melanocytes by Targeting Wnt3a
Sulindac has strong antifibrotic effects by suppressing STAT3-related miR-21
Adenovirus-mediated transfection with glucose transporter 3 suppresses PC12 cell apoptosis following ischemic injury
(?)-Epigallocatechingallate induces apoptosis in B lymphoma cells via caspase-dependent pathway and Bcl-2 family protein modulation
Down-regulation of Homer1b/c expression protects cultured neurons after traumatic injury
Cisplatin upregulates MSH2 expression by reducing miR-21 to inhibit A549 cell growth
Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation
PLK1 gene suppresses cell invasion of undifferentiated thyroid carcinoma through the inhibition of CD44v6, MMP-2 and MMP-9
siRNA targeting decoy receptor 3 enhances the sensitivity of gastric carcinoma cells to 5-fluorouracil
Effect of essential fatty acids on glucose-induced cytotoxicity to retinal vascular endothelial cells
A single residue change in Vibrio harveyi hemolysin results in the loss of phospholipase and hemolytic activities and pathogenicity for turbot (Scophthalmus maximus).
Effect of Buddleia flavonoids drug-containing plasma on the expression of STAT1 phosphoprotein in lacrimal gland epithelial cells in vitro
RNAi-mediated silencing of CD147 inhibits tumor cell proliferation, invasion and increases chemosensitivity to cisplatin in SGC7901 cells in vitro
A single residue change in Vibrio harveyi hemolysin results in the loss of phospholipase and hemolytic activities and pathogenicity for turbot (Scophthalmus maximus).
Effect of Buddleia flavonoids drug-containing plasma on the expression of STAT1 phosphoprotein in lacrimal gland epithelial cells in vitro
RNAi-mediated silencing of CD147 inhibits tumor cell proliferation, invasion and increases chemosensitivity to cisplatin in SGC7901 cells in vitro
Xue D, Xue Y, Niu Z, Guo X, Xu C. Genet Mol Biol. 2017 Oct-Dec;40(4):855-859. doi: 10.1590/1678-4685-GMB-2017-0029. Epub 2017 Nov 6. Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy
Guo X, Fu Y, Wang Z, Wang T, Li C, Huang T, Gao F, Li C. Oxid Med Cell Longev. 2018 Mar 25;2018:4950705. doi: 10.1155/2018/4950705. eCollection 2018. Di-2-pyridylhydrazone Dithiocarbamate Butyric Acid Ester Exerted Its Proliferative Inhibition against Gastric Cell via ROS-Mediated Apoptosis and Autophagy
Li M, Zhou J, Jin W, Li X, Zhang Y. Front Pharmacol. 2018 Apr 6;9:308. doi: 10.3389/fphar.2018.00308. eCollection 2018. Danhong Injection Combined With t-PA Improves Thrombolytic Therapy in Focal Embolic Stroke
Wang J, Guo XJ, Ding YM, Jiang JX. World J Gastroenterol. 2017 Mar 7;23(9):1594-1601. doi: 10.3748/wjg.v23.i9.1594. miR-1181 inhibits invasion and proliferation via STAT3 in pancreatic cancer
Mei ZG, Tan LJ, Wang JF, Li XL, Huang WF, Zhou HJ. Neural Regen Res. 2017 Mar;12(3):425-432. doi: 10.4103/1673-5374.202946. Fermented Chinese formula Shuan-Tong-Ling attenuates ischemic stroke by inhibiting inflammation and apoptosis
Wu M1, Huang C2, Huang X1, Liang R3, Feng Y1, Luo X1. Oncol Rep. 2017 Oct;38(4):2173-2181. doi: 10.3892/or.2017.5900. Epub 2017 Aug 11. MicroRNA-144-3p suppresses tumor growth and angiogenesis by targeting SGK3 in hepatocellular carcinoma
Yan J, Wang J, Huang H, Huang Y, Mi T, Zhang C, Zhang L. Am J Transl Res. 2017 Oct 15;9(10):4492-4501. eCollection 2017. Fibroblast growth factor 21 delayed endothelial replicative senescence and protected cells from H2O2-induced premature senescence through SIRT1
Liu B, Liu C, Cong W, Li N, Zhou N, Tang Y, Wei C, Bai H, Zhang Y, Xiao J. Skelet Muscle. 2017 May 11;7(1):8. doi: 10.1186/s13395-017-0126-x. Retinoid acid-induced microRNA-31-5p suppresses myogenic proliferation and differentiation by targeting CamkIIδ
Wang R, Shen Z, Yang L, Yin M, Zheng W, Wu B, Liu T, Song H. Int J Mol Med. 2017 Nov;40(5):1537-1548. doi: 10.3892/ijmm.2017.3121. Epub 2017 Sep 6. Protective effects of heme oxygenase-1-transduced bone marrow-derived mesenchymal stem cells on reduced-size liver transplantation: Role of autophagy regulated by the ERK/mTOR signaling pathway
Feng XE, Liang TG, Gao J, Kong P, Ge R, Li QS,. Oxid Med Cell Longev. 2017;2017:7028478. doi: 10.1155/2017/7028478. Epub 2017 Jun 14. Heme Oxygenase-1, a Key Enzyme for the Cytoprotective Actions of Halophenols by Upregulating Nrf2 Expression via Activating Erk1/2 and PI3K/Akt in EA.hy926 Cells
Tang S, Pan H, Wei W, Yang H, Liu J, Yang R. Oncotarget. 2017 Oct 19;8(62):105155-105169. doi: 10.18632/oncotarget.21927. eCollection 2017 Dec 1. GOLPH3: a novel biomarker that correlates with poor survival and resistance to chemotherapy in breast cancer
Wu YX, Sun Y, Ye YP, Zhang P, Guo JC, Huang JM, Jing XZ, Xiang W, Yu SY, Guo FJ. Mol Med Rep. 2017 Dec;16(6):8200-8208. doi: 10.3892/mmr.2017.7648. Epub 2017 Sep 28. Iguratimod prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator-activated receptor-γ
Huang JM, Bao Y, Xiang W, Jing XZ, Guo JC, Yao XD, Wang R, Guo FJ. Evid Based Complement Alternat Med. 2017;2017:8085325. doi: 10.1155/2017/8085325. Epub 2017 Dec 27. Icariin Regulates the Bidirectional Differentiation of Bone Marrow Mesenchymal Stem Cells through Canonical Wnt Signaling Pathway
Guo J, Li W, Wu Y, Jing X, Huang J, Zhang J, Xiang W, Ren R, Lv Z, Xiao J, Guo F. Front Pharmacol. 2017 Oct 4;8:693. doi: 10.3389/fphar.2017.00693. eCollection 2017. Meclizine Prevents Ovariectomy-Induced Bone Loss and Inhibits Osteoclastogenesis Partially by Upregulating PXR
Li YF, Yuan YY, Zhang YM, Zhao N, Zhang Q, Meng FX, Gao RP, Yu BF, Zhang YH, Guo R, Wang HL, Xie J, Xu J, Qin Q, Dong XS. Mol Med Rep. 2017 Jul;16(1):764-772. doi: 10.3892/mmr.2017.6657. Epub 2017 May 31. HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1-pAFP-TK and targeted killing effect
Liang B, Xiao T, Long J, Liu M, Li Z, Liu S, Yang J. Int J Mol Med. 2017 Dec;40(6):1781-1791. doi: 10.3892/ijmm.2017.3191. Epub 2017 Oct 16. Hydrogen sulfide alleviates myocardial fibrosis in mice with alcoholic cardiomyopathy by downregulating autophagy
Chen W, Liu X, Yuan S, Qiao T. Oncol Lett. 2018 Mar;15(3):3883-3889. doi: 10.3892/ol.2018.7800. Epub 2018 Jan 16. HSPA12B overexpression induces cisplatin resistance in non-small-cell lung cancer by regulating the PI3K/Akt/NF-κB signaling pathway
Bai J, Yang BJ, Luo X. Oncol Lett. 2018 Apr;15(4):5227-5235. doi: 10.3892/ol.2018.7938. Epub 2018 Feb 2. Effects of 5-hydroxy-4
Yuan X, Zhang Z, Jiang K, Wang X, Li Y. Med Sci Monit. 2018 Feb 21;24:1080-1088. Preliminary Study of the Role F-Box Protein 32 (FBXO32) in Colorectal Neoplasms Through the Transforming Growth Factor beta (TGF-β)/Smad4 Signalling Pathway
Kai Xu, Qingsheng Niu, Huiting Zhao, Yali Du, Lina Guo, and Yusuo Jiang. Journal of Insect Science. Sequencing and Expression Characterization of Antifreeze Protein Maxi-Like in Apis cerana cerana.
Wang P, Mao Z, Pan Q, Lu R, Huang X, Shang X, Zhang R, You H. Int J Mol Med. 2018 Apr;41(4):2117-2127. doi: 10.3892/ijmm.2018.3410. Epub 2018 Jan 22. Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways.
Ma, Z., Cai, H., Zhang, Y., Chang, L., & Cui, Y. (2017). MiR-129-5p inhibits non-small cell lung cancer cell stemness and chemoresistance through targeting DLK1. Biochemical and Biophysical Research Communications. Advance online publication. doi: 10.1016/j.bbrc.2017.06.041