Affinity ECL kit (femtogram) - KF003

单价 规格
¥1500 100ml
¥3600 500ml

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  • 名 称:
    Affinity ECL kit (femtogram)
  • 货 号:
    KF003
  • 应 用:
    WB

产品信息

 Affinity® ECL Western Blotting Substrate (fg grade)

Introduction

The Affinity ECL Western Blotting Substrate is a highly sensitive nonradioactive, enhanced luminol-based chemiluminescent substrate for the detection of horseradish peroxidase (HRP) on immunoblots. Affinity ECL Western Blotting Substrate enables the detection of femtogram amounts of antigen and allows for easy detection of HRP using photographic or other imaging methods. Blots can be repeatedly exposed to X-ray film to obtain optimal results or stripped of the immunodetection reagents and re-probed. The special formulation of Affinity ECL Substrate makes it the ideal substitute for other ECL Substrate without the need for additional optimization of assay conditions.

 

Important Product Information    

Affinity ECL Substrate requires more dilute antibody concentrations than those used with precipitating colorimetric HRP substrates. To optimize antibody concentrations, perform a systematic dot blot analysis.

• Empirical testing is essential to determine the appropriate blocking reagent for each Western blot system, as crossreactivity of the blocking reagent with antibodies causes nonspecific signal. Blocking buffer also affects system sensitivity.  

• Avoid using milk as a blocking reagent when using avidin/biotin systems because 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 blot and ensure that it never becomes dry. Using large blocking and wash buffer volumes minimizes nonspecific signal.

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

• 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 sodium azide as a preservative for buffers. Sodium azide is an inhibitor of HRP and could interfere with this system.

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

• All equipment must be clean and free of foreign material. Metallic devices (e.g., scissors) 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.  

 

Procedure Summary  

Note: Antigen and antibody amounts may require optimization.  

1. Dilute the primary antibody to 5.0-0.5µg/mL.  

2. Dilute the secondary antibody to 1.0-0.067µg/mL.  

3. Mix Detection Reagents 1 and 2 at a 1:1 ratio and add it to the blot. Incubate blot for 1-60 seconds.

4. Drain excess reagent. Cover blot with a clear plastic sheet protector or clear plastic wrap.

5. Expose blot to X-ray film.

 

Detailed Western Blotting Procedure 

1. Remove blot from the transfer apparatus and block nonspecific sites with Blocking Reagent for 60 minutes at room temperature (RT) with shaking. If desired, block overnight at 2-8ºC without shaking.

2. Remove the Blocking Reagent and add the primary antibody working dilution. Incubate blot for 1 hour at RT with shaking or overnight at 2-8°C without shaking.

3. Briefly rinse membrane in Wash Buffer two times.

4. Wash membrane by suspending it in Wash Buffer and agitating for ≥ 5 minutes. Replace Wash Buffer at least 4-6 times. Increasing the Wash Buffer volume, the number of washes and wash duration may help minimize background signal.

5. Incubate blot with the HRP-conjugate working dilution for 1 hour at RT with shaking.

6. Repeat Steps 3 and 4 to remove nonbound HRP-conjugate.

Note: Membrane MUST be thoroughly washed after incubation with the HRP-conjugate.

7. Prepare the substrate working solution by mixing equal parts of Detection Reagents 1 and 2. Use 0.125mL Working Solution per cm2 of membrane.  

Note: For best results prepare working solution immediately before use. The working solution is stable for 1 hour at RT.  

8. Incubate blot with working solution for 1 minute at RT.

9. Remove blot from working solution and place it in a plastic sheet protector or clear plastic wrap. Use an absorbent tissue to remove excess liquid and to 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. Turn off all lights except those appropriate for X-ray film exposure (e.g., a red safelight).

Note: Film must remain dry during exposure. For optimal results, perform the following precautions:

• Make sure excess substrate is removed from the membrane and the membrane protector.

• Use gloves during the entire film-handling process.

• Never place a blot on developed film, as chemicals on the film may reduce signal.

11. Carefully place X-ray film on top of the membrane. Perform a first-time exposure of 30 seconds. Vary the exposure time to achieve optimal results.  

12. Develop film using appropriate developing solution and fixative. If signal is too intense, reduce exposure time or strip and re-probe the blot with decreased antibody concentrations.  

 

Storage

Upon receipt store at 2-8°C.

 

Cat.#

Product Name

Size

K003

Solution

25/50/250 ml

K004

Solution

25/50/250 ml

 

 

Affinity®ECL Reagent,20s exposure time under a variety of dilutions. The secondary antibody is Goat anti-rabbit IgG-Hrp(Cat.# S0001).

Reference Citations:

1). Wang K;Wu Q;Wang X;Liang G;Yang A;Li J; et al. Near-infrared control and real-time detection of osteogenic differentiation in mesenchymal stem cells by multifunctional upconversion nanoparticles† . Nanoscale 2020 May 14;12(18):10106-10116. (PubMed: 32350492)

Application: ;   Species:Not available;    Sample:Not available;



2). Liang C et al. Fluoride induced mitochondrial impairment and PINK1-mediated mitophagy in Leydig cells of mice: In vivo and in vitro studies. Environ Pollut 2020 Jan;256:113438 (PubMed: 31672359)

Application: ;   Species:Not available;    Sample:Not available;



3). Sun W et al. Adipogenic activity of 2-ethylhexyl diphenyl phosphate via peroxisome proliferator-activated receptor γ pathway. Sci Total Environ 2020 Apr 1;711:134810 (PubMed: 31812418)

Application: ;   Species:Not available;    Sample:Not available;



4). Lai N et al. Systemic exosomal miR-193b-3p delivery attenuates neuroinflammation in early brain injury after subarachnoid hemorrhage in mice. J Neuroinflammation 2020 Feb 25;17(1):74 (PubMed: 32098619)

Application: ;   Species:Not available;    Sample:Not available;



5). Zhou L et al. Carbon black nanoparticles induce pulmonary fibrosis through NLRP3 inflammasome pathway modulated by miR-96 targeted FOXO3a. Chemosphere 2020 Feb;241:125075 (PubMed: 31683435)

Application: ;   Species:Not available;    Sample:Not available;



6). Li M;Cao J;Zhao Y;Wu P;Li X;Khodaei F;Han Y;Wang J; et al. Fluoride impairs ovary development by affecting oogenesis and inducing oxidative stress and apoptosis in female zebrafish (Danio rerio). Chemosphere 2020 May 17;256:127105. (PubMed: 32450357)

Application: ;   Species:Not available;    Sample:Not available;



7). Yang L et al. Neuroprotection by dihydrotestosterone in LPS-induced neuroinflammation. Neurobiol Dis 2020 Feb 19:104814 (PubMed: 32087283)

Application: ;   Species:Not available;    Sample:Not available;



8). Liu N;Sun Q;Xu H;Yu X;Chen W;Wei H;Jiang J;Xu Y;Lu W; et al. Hyperuricemia induces lipid disturbances mediated by LPCAT3 upregulation in the liver. FASEB J 2020 Aug 11. (PubMed: 32780898)

Application: ;   Species:Not available;    Sample:Not available;



9). Zhou L et al. PM2.5 exposure impairs sperm quality through testicular damage dependent on NALP3 inflammasome and miR-183/96/182 cluster targeting FOXO1 in mouse. Ecotoxicol Environ Saf 2019 Mar;169:551-563 (PubMed: 30476817)

Application: ;   Species:Not available;    Sample:Not available;



10). Zhang D et al. Wnt-3a alleviates neuroinflammation after ischemic stroke by modulating the responses of microglia/macrophages and astrocytes. Int Immunopharmacol 2019 Jul 16;75:105760 (PubMed: 31323530)

Application: ;   Species:Not available;    Sample:Not available;



11). Wang X;Yin H;Fan L;Zhou Y;Tang X;Fei X;Tang H;Peng J;Zhang J;Xue Y;Luo J;Jin Q;Jin Q; et al. Shionone alleviates NLRP3 inflammasome mediated pyroptosis in interstitial cystitis injury. Int Immunopharmacol 2020 Nov 19;107132. (PubMed: 33223465)

Application: ;   Species:Not available;    Sample:Not available;



12). Liu L et al. Melatonin ameliorates cerebral ischemia/reperfusion injury through SIRT3 activation. Life Sci 2019 Nov 4:117036 (PubMed: 31697951)

Application: ;   Species:Not available;    Sample:Not available;



13). Zhou J et al. MicroRNA-145 overexpression attenuates apoptosis and increases matrix synthesis in nucleus pulposus cells. Life Sci 2019 Feb 20 (PubMed: 30797016)

Application: ;   Species:Not available;    Sample:Not available;



14). Lu Y et al. The Role of MKP-5 in Adipocyte-Macrophage Interactions during Obesity. Obes Facts 2020 Jan 21:1-16 (PubMed: 31962332)

Application: ;   Species:Not available;    Sample:Not available;



15). Li M;Feng L;Jiang WD;Wu P;Liu Y;Jiang J;Kuang SY;Tang L;Zhou XQ; et al. Betaine supplementations enhance the intestinal immunity of on-growing grass carp (Ctenopharyngodon idella): partly related to TOR and NF-κB signaling pathways . Br J Nutr 2020 Apr 14;123(7):737-755. (PubMed: 31831090)

Application: ;   Species:Not available;    Sample:Not available;



16). Li M;Feng L;Jiang WD;Wu P;Liu Y;Jiang J;Kuang SY;Tang L;Zhou XQ; et al. Betaine supplementations enhance the intestinal immunity of on-growing grass carp (Ctenopharyngodon idella): partly related to TOR and NF-κB signaling pathways . Br J Nutr 2020 Apr 14;123(7):737-755. (PubMed: 31831090)

Application: ;   Species:Not available;    Sample:Not available;



17). Peng XR et al. Supplementation exogenous bile acid improved growth and intestinal immune function associated with NF-κB and TOR signalling pathways in on-growing grass carp (Ctenopharyngodon idella): Enhancement the effect of protein-sparing by dietary lipid. Fish Shellfish Immunol 2019 Jun 26;92:552-569 (PubMed: 31252043)

Application: ;   Species:Not available;    Sample:Not available;



18). Zheng L et al. Selenium deficiency impaired structural integrity of the head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol 2018 Nov;82:408-420 (PubMed: 30142391)

Application: ;   Species:Not available;    Sample:Not available;



19). Zhong JR et al. Phytic acid disrupted intestinal immune status and suppressed growth performance in on-growing grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol 2019 Jun 24;92:536-551 (PubMed: 31247320)

Application: ;   Species:Not available;    Sample:Not available;



20). Huang C et al. Deoxynivalenol decreased intestinal immune function related to NF-κB and TOR signalling in juvenile grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol 2018 Oct 16;84:470-484 (PubMed: 30339843)

Application: ;   Species:Not available;    Sample:Not available;



21). Zhao Y et al. Fine particulate matter (PM2.5) enhances airway hyperresponsiveness (AHR) by inducing necroptosis in BALB/c mice. Environ Toxicol Pharmacol 2019 May;68:155-163 (PubMed: 30986632)

Application: ;   Species:Not available;    Sample:Not available;



22). Wang Z;Cui M;Qu Y;He R;Wu W;Lin H;Shao Z; et al. Hypoxia Protects Rat Bone Marrow Mesenchymal Stem Cells Against Compression-Induced Apoptosis in the Degenerative Disc Microenvironment Through Activation of the HIF-1α/YAP Signaling Pathway. Stem Cells Dev 2020 Sep 9. (PubMed: 32799744)

Application: ;   Species:Not available;    Sample:Not available;



23). Zhao J et al. Down-regulation of long noncoding RNA DLX6-AS1 defines good prognosis and inhibits proliferation and metastasis in human epithelial ovarian cancer cells via Notch signaling pathway. Eur Rev Med Pharmacol Sci 2019 Apr;23(8):3243-3252 (PubMed: 31081076)

Application: ;   Species:Not available;    Sample:Not available;



24). Liu W et al. Dynamic characterization of intestinal metaplasia in the gastric corpus mucosa of Atp4a-deficient mice. Biosci Rep 2020 Jan 6 (PubMed: 31904088)

Application: ;   Species:Not available;    Sample:Not available;



25). Zhang J et al. The immunogenicity and protective immunity of multi-epitopes DNA prime-protein  boost vaccines encoding Amastin-Kmp-11, Kmp11-Gp63 and Amastin-Gp63 against visceral leishmaniasis. PLoS One 2020 Mar 16;15(3):e0230381 (PubMed: 32176727)

Application: ;   Species:Not available;    Sample:Not available;



26). Zhang J;Hou C;Dou S;Li G;Wang Z;Liu Y;Zhang Y;Wang R;Shen B;Han G; et al. T cell immunoglobulin and mucin domain protein 3 inhibits glycolysis in RAW 264.7 macrophages through Hexokinase 2. Scand J Immunol 2020 Oct 8;e12981. (PubMed: 33031600)

Application: ;   Species:Not available;    Sample:Not available;



27). Liang T;Ma C;Wang T;Deng R;Ding J;Wang W;Xu Z;Li X;Li H;Sun Q;Shen H;Wang Z;Chen G; et al. Galectin-9 Promotes Neuronal Restoration via Binding TLR-4 in a Rat Intracerebral Hemorrhage Model. Neuromolecular Med 2020 Aug 31. (PubMed: 32865657)

Application: ;   Species:Not available;    Sample:Not available;



28). Ruan H et al. Anlotinib attenuated bleomycin-induced pulmonary fibrosis via the TGF-β1 signalling pathway. J Pharm Pharmacol 2019 Oct 28 (PubMed: 31659758)

Application: ;   Species:Not available;    Sample:Not available;



29). Kan J;Huang H;Jiang Z;Zhou R;Bai S;Liao C;Chen J;Dong J;Zhang Y;Zhang J;Zhang R;Zhou D;Zhang E; et al. Arenobufagin Promoted Oxidative Stress-Associated Mitochondrial Pathway Apoptosis in A549 Non-Small-Cell Lung Cancer Cell Line. Evid Based Complement Alternat Med 2020 Apr 22;2020:8909171. (PubMed: 32382311)

Application: ;   Species:Not available;    Sample:Not available;



30). Zhao L;Yang R;Cheng L;Wang M;Jiang Y;Wang S; et al. Role of TGF‑β1‑mediated epithelial‑mesenchymal transition in the pathogenesis of tympanosclerosis. J Korean Med Sci 2010 Jul;25(7):1066-70. (PubMed: 20592900)

Application: ;   Species:Not available;    Sample:Not available;



31). Chen M et al. The Therapeutic Effects and Possible Mechanism of Pranoprofen in Mouse Model of Corneal Alkali Burns. J Ophthalmol 2020 Apr 6;2020:7485912 (PubMed: 32322412)

Application: ;   Species:Not available;    Sample:Not available;



32). Liu X et al. Nck1 promotes the progression of ovarian carcinoma by enhancing the PI3K/AKT/p70S6K signaling. Hum Cell 2020 Mar 12 (PubMed: 32166565)

Application: ;   Species:Not available;    Sample:Not available;



33). et al. Evaluation of curcumin-mediated photodynamic therapy on the reverse of multidrug resistance in tumor cells.

Application: ;   Species:Not available;    Sample:Not available;



34). et al. Salidroside Attenuates Cognitive Dysfunction in Senescence-Accelerated Mouse Prone 8 (SAMP8) and Modulating Inflammation on the Gut-brain Axis.

Application: ;   Species:Not available;    Sample:Not available;



35). Wu Q et al. Delivering siRNA to control osteogenic differentiation and real-time detection of cell differentiation in human mesenchymal stem cells using multifunctional gold nanoparticles. J Mater Chem B 2020 Apr 21;8(15):3016-3027 (PubMed: 32207489)

Application: ;   Species:Not available;    Sample:Not available;



36). et al. CircRHOT1 Mediated Cell Proliferation, Apoptosis and Invasion of Pancreatic Cancer Cells by Sponging miR125a-3p.

Application: ;   Species:Not available;    Sample:Not available;



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