After rinsing with PBS, the cells were lysed in ice for 30 min in lysis buffer (Beyotime Institute of Biotechnology) containing a cocktail protease inhibitor (Roche). After centrifuging at 12,000 g for 15 min, the supernatant was extracted and saved at −80°C. Protein samples were electrophoresed on a 10% sodium dodecyl sulfate gel and transferred to polyvinylidene difluoride membrane at 300 mA for 2 h. The blots were blocked in blocking buffer (0.1% Tween-20 in PBS containing 5% non-fat dry milk, pH 7.4) for 2 h. Afterward, the membrane was incubated overnight with the corresponding antibodies in blocking buffer at 4°C: LRG1 antibody (HPA001888, rabbit polyclonal; Sigma, St Louis, MO, USA) or Actin antibody (sc-1616, goat polyclonal; Santa Cruz Biotechnology, Dallas, TX, USA). After washing with PBST thrice, the blots were incubated with secondary antibody (1:40,000) (Santa Cruz Biotechnology) in PBST for 2 h at room temperature. The blots were washed again thrice in PBST buffer and transferred proteins were detected with an enhanced chemiluminescence detection kit (Applygen, Beijing, China).
Enhanced chemiluminescence detection kit
Manufactured by Applygen
Sourced in China
The Enhanced Chemiluminescence Detection Kit is a laboratory equipment designed to detect and quantify proteins in biological samples. It utilizes the principle of chemiluminescence, where a light-emitting chemical reaction occurs when specific reagents interact with the target proteins. This kit provides the necessary components to perform sensitive and reliable protein detection assays.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Quantity one image analyzer software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Applygen (1 mentions)
Protein extraction kit, by Applygen (1 mentions)
Protease inhibitor cocktail, by Avantor (1 mentions)
Nonfat milk, by BD (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Wet transblot system, by Bio-Rad (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 mini system, by GE Healthcare (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Applygen (1 mentions)
Anti sting, by Cell Signaling Technology (1 mentions)
Anti ifn γ, by ABclonal (1 mentions)
Anti p tbk1, by Cell Signaling Technology (1 mentions)
Anti il 17a, by Bioworld Technology (1 mentions)
Anti p sting, by Cell Signaling Technology (1 mentions)
7 protocols using enhanced chemiluminescence detection kit
1
Quantitative Protein Analysis by Western Blot
2
Protein Extraction and Western Blot Analysis
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Using a protein extraction kit (Applygen Technologies), total protein was extracted and the concentration of protein was determined by a BCA protein assay kit (Applygen Technologies). Western blot analysis was performed routinely (10 (link)), with primary antibodies against NDUFA10 (Santa Cruz, Santa Cruz, Calif), adenosine triphosphate synthase δ-subunit (ATP 5D) (Abcam), Sirt1, Sirt3, SDHA, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, Mass). Enhanced chemiluminescence detection kit (Applygen Technologies, Beijing, China) was used to detect the bands. Band intensity was expressed as mean area density using Quantity One image analyzer software (Bio-Rad, Richmond, Calif) for quantification.
3
Western Blot Analysis of Paclitaxel Resistance
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Paclitaxel-resistant cells were lysed in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride and EDTA) containing protease inhibitor cocktails (Amresco, Solon, OH, USA). Equal amounts of cell lysates were resolved by SDS-PAGE and subsequently electrophoretically transferred to PVDF membranes (Millipore, Darmstadt, Germany). After blocking in TBST (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween 20, pH 8.0) with 5% non-fat milk (BD Biosciences, Franklin Lakes, NJ, USA) for 2 h at room temperature, the membranes were incubated with primary and secondary antibodies and subsequently visualized with an enhanced chemiluminescence detection kit (APPLYGEN Technologies Inc., Beijing, China). Actin was used as a loading control.
4
Western Blot Analysis of TRIM68 in SW1116 Cells
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Prior to applying western blot analysis, SW1116 cells were subcultured and infected at 37°C with recombinant lentiviruses for 4 days. The cells were rinsed with cold PBS twice, and tsubsequently lysed in cell lysis buffer containing 10 mM EDTA, 4% SDS, 10% glycine in 100 mM Tris-HCl (pH 6.8) (Shanghai Hollybio Co., Ltd.). Equal amounts of protein samples (60 µg) were subjected to 10% SDS-PAGE in a Tris/HCl buffer (pH 7.4) and electrotransferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA) at 300 mA for 1.5 h. The membrane was blocked for 1 h in PBST buffer (PBS pH 7.4, 0.05% Triton-100) containing 1% (w/v) bovine serum albumin at room temperature. The membrane was then probed with a rabbit anti-TRIM68 antibody (dilution, 1:500; cat. no. AP17177b; Abgent Biotech Co., Ltd., Suzhou, China) and rabbit anti-GAPDH antibody simultaneously (dilution, 1:3,000; cat. no. 10494-1-AP; Proteintech Group, Inc., Chicago, IL, USA) overnight at 4°C, followed by 5 washes with PBST. Blots were then incubated with a secondary goat anti-rabbit horseradish peroxidase-conjugated antibody (dilution, 1:5,000; cat. no. SC-2054; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit (Applygen Technologies, Inc., Beijing, China) in accordance with the manufacturer's protocol.
5
Protein Extraction and Western Blot Analysis
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Cells were harvested and total protein was extracted using radioimmunoprecipitation assay lysis buffer (catalog number C1053; Applygen, Beijing, China) supplemented with protease inhibitor cocktail (catalog number 78410; Thermo Fisher Scientific). The Pierce BCA Protein Assay Kit was used to quantify the protein concentration (catalog number 23225; Thermo Fisher Scientific). Subsequently, equal amounts of the proteins were separated by SDS-PAGE and transferred onto a 0.25-mm polyvinylidene difluoride membrane (catalog number IPFL00010; Millipore, Burlington, MA) via a wet trans-blot system (BioRad, Hercules, CA). The membranes were then blocked and incubated with an antibody against NCAPD2 (catalog number ab56885; Abcam, Cambridge, UK) overnight at 4 C, followed by incubation with a horseradish peroxidaseeconjugated secondary antibody (catalog number ZDR-5307; Zhongshan Golden Bridge Biotechnology Company) and analyzed using an enhanced chemiluminescence detection kit (catalog number P1050; Applygen). Finally, images were obtained with an ImageQuant LAS 4000 mini system (GE Healthcare, Chicago, IL). b-actin (catalog number sc-58673; Santa Cruz Biotechnology, Santa Cruz, CA) was used as an internal control.
6
Western Blot Analysis of Oral Lichen Planus
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Fresh tissue samples from patients with OLP and healthy controls were carefully dissected. Protein concentrations were measured according to a BCA assay (Thermo Scientifc, Waltham, MA, USA) generated standard curve. A total of 40 µg of protein was denatured and then subjected to 12% SDS-polyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene fuoride membranes (Millipore Corporation, Billerica, MA, USA). The membranes were incubated with primary antibodies (anti-γδ TCR (Thermo Fisher, Catalog #5A6.E9), anti-STING (Cell Signaling Technology, Catalog #D2P2F), anti-NAK/TBK1 (Abcam, Catalog #EP611Y), anti-p-STING (Cell Signaling Technology, Catalog #E9A9K), anti-p-TBK1 (Cell Signaling Technology, Catalog #D52C2), anti-IFN-γ (Abclonal, Wuhan, China, Catalog #A12450), anti-IL-17A (Bioworld, Dublin, OH, USA, Catalog #BS6041), anti-HLA-DR (Nonus, CO, USA, Catalog #NBP2-67610) and anti-CCR6 (Bioss, Beijing, China, Catalog #BS-1542R)). After being washed, the membranes were then probed with secondary antibodies. Next, the blots were stained using an enhanced chemiluminescence detection kit (Applygen, Beijing, China). The relative protein levels were calculated based on β-actin (Proteintech, Chicago, IL, USA, Catalog #66009-1-Ig) as the loading control and were densitometrically analyzed by Image J software (NIH, Bethesda, MD, USA).
7
Protein Extraction and Western Blot Analysis
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Total protein was extracted using radioimmunoprecipitation assay lysis buffer and the protein concentration was determined using a bicinchoninic acid assay (Sigma-Aldrich). Subsequently, 50 mg total protein was resolved using SDS-PAGE and the separated protein was transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked in 5% non-fat milk for 1 h and then incubated with primary antibodies overnight at 4°C. After washing three times with phosphate-buffered saline with Tween-20, the membrane was incubated with peroxidase-conjugated AffiniPure goat anti-mouse (ZB-2305) and goat anti-rabbit (ZB-2301) immunoglobulin G secondary antibodies (1:3,000; Zhongshan Jinqiao Biotech, Co., Ltd., Beijing, China) for 1 h at room temperature. Bands were detected with an enhanced chemiluminescence detection kit (Applygen Technologies, Inc., Beijing, China) and the densitometry of each band was analyzed with ImageJ software.
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