We used 1% PMSF and RIPA lysine buffer to confine cellular protein, then reacted with the SDS-PAGE test buffer. Proteins were transferred to a polyvinylide difluoride layer (Millipur, USA). The layer was grown overnight, after incubation for 1 h, at room temperature. Then we applied the ECL chemiluminescence kit (Advansta, USA), and proteins were fried with secondary antibodies for 1 h. We examined bands using GeneGnome 5 (Synoptics Ltd., UK).
Ecl chemiluminescence kit
Manufactured by Advansta
Sourced in United States
The ECL chemiluminescence kit is a laboratory equipment used for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent substrate that emits light upon exposure to the enzyme-labeled target proteins, allowing for sensitive and accurate protein visualization.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Zc3h13, by Abcam (1 mentions)
Ythdf2, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Bca kit, by Solarbio (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Dingguo (1 mentions)
Ma1 16757, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 plus goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
7 protocols using ecl chemiluminescence kit
1
Protein Extraction and Western Blot Analysis
2
Amyloid Oligomers and Fibril Detection
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Incubated samples were prepared as described. 3 μL of sample solution aliquoted at indicated time points was blotted onto a nitrocellulose membrane (Biorad, USA). Dried membrane was blocked with 5% non-fat milk for 1 h at room temperature and then incubated with A-11 (oligomer-specific antibody; 1:2500) or OC antibody (fibril-specific antibody; 1:2500) at 4°C overnight. The membrane was incubated with anti-rabbit IgG (1:5000) for 2 h at room temperature later. An ECL chemiluminescence kit (Advansta, USA) was used for the final blot development.
3
Protein Expression Analysis by Western Blot
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Cellular protein in three distinctive groups was confined by 1% PMSF and RIPA lysis buffer (50mM Tris-HCl (pH7.4), 150mM NaCl, 1%NP-40, 0.1% SDS). After reacted with SDS-PAGE test buffer, sodium dodecy lsulfate–polyacrylamide gel electrophoresis was used to perform further examination. At that point, the proteins were exchanged onto a polyvinylidene difluoride layer (Millipore, USA). After being blocked for 1h at room temperature, the layer was brooded with anti-Rabbit TRIM29 (1:1000) (#5182, CST, USA), GAPDH (1:1000) (#2118, CST, USA), E-Cadherin (1:1000) (#31958, CST, USA), LC3-I (1:1000) (#4599, CST, USA), LC3-II (1:1000) (#3868, CST, USA), p62 (1:1000) (#16177, CST, USA), and BECN1(1:1000) (#14717, CST, USA) overnight. Proteins were hatched with the corresponding secondary antibodies for 1 h at room temperature after treated with ECL chemiluminescence kit (Advansta, USA). The bands were observed with GeneGnome 5 (Synoptics Ltd., UK).
4
Immunoblotting Oligomers and Fibrils
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Sample aliquotes (2 μL) obtained at indicated time points were blotted onto a nitrocellulose membrane (Bio-Rad, USA). Dried membrane was blocked with 5% non-fat milk for 1 h at room temperature and then incubated with anti-oligomer antibody (A-11) or anti-fibril antibody (OC) at 4 °C overnight. The membrane was incubated with anti-rabbit IgG for 2 h at room temperature later. An ECL chemiluminescence kit (Advansta, USA) was used for the development.
5
Western Blot and qRT-PCR Analysis of YTHDF2 and ZC3H13
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RIPA buffer containing the protease inhibitor PMSF (Solarbio Science & Technology Company, China) was used to lyse tissues on ice, and BCA kit (Solarbio Science & Technology Company, China) was used for protein quantification. A total of 20 µg proteins were separated by 10% SDS-PAGE and electro-blotted onto nitrocellulose (NC) membrane. After sealing with skimmed milk, the NC membrane was incubated with the first antibody at 4 °C overnight. The membranes were washed and incubated with the second antibody on the shaking table at room temperature for two hours. ECL chemiluminescence kit (Advansta, USA) was used to visualize the protein bands. β-actin was used as a control. The main antibodies used in this study included YTHDF2 (1:1,000) and ZC3H13 (1:1,000) (Abcam, USA).
For mRNA quantifications of YTHDF2 and ZC3H13, cDNA was synthesized by DNase treatment and reverse transcription (TIANGEN Biotech Company, China). Real-time PCR was on TL988 Real-Time PCR Detection System (TIANLONG, China). The primers were listed inTable 1 . The mRNA levels of the selected genes were normalized to that of the reference gene β-actin, and the value were calculated by the 2−ΔΔCt method. The results are expressed as the means ± standard error based on three independent experiments.
For mRNA quantifications of YTHDF2 and ZC3H13, cDNA was synthesized by DNase treatment and reverse transcription (TIANGEN Biotech Company, China). Real-time PCR was on TL988 Real-Time PCR Detection System (TIANLONG, China). The primers were listed in
6
Protein Extraction and Western Blotting
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RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with 1% phenylmethylsulfonyl fluoride (PMSF, Dingguo, Beijing, China) was used for protein extraction of cells and tissues. The protein concentration was measured with a BCA kit (Dingguo, Beijing, China) according to the manufacturer’s instructions. Protein samples (40 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with TBST containing 5% skimmed milk at room temperature for 1.5 hours and incubated with specific primary antibody diluted with 5% BSA at 4°C overnight. After incubation with secondary antibody for 2 hours at room temperature, the protein signals were detected using a ECL chemiluminescence kit (Advansta, San Jose, CA, USA). Immunoprecipitation was performed as previously described (34 (link)). Grayscale value analysis of all protein bands was performed via ImageJ 1.46r software (NIH, Bethesda, MD, USA).
7
Western Blot Protein Analysis
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Protein from tissues or cells was released by using RIPA lysis buffer. We measured the protein concentration by BCA protein assay kit (Bio-Rad, USA). 20 mg protein was obtained by using SDS-PAGE gels. Then, the extracted protein was moved by electricity to a PVDF membrane (Millipore, Bedford, MA, USA), then, interacted with specific primary antibody anti-APITD1 (1 : 1000; #PA5-24846, RRID : AB_2542346; Invitrogen, Shanghai, China) or anti-GAPDH (1 : 1000; #MA1-16757, RRID : AB_568547; Invitrogen, Shanghai, China) overnight after being treated with 5% fat-free milk solution for 60 min at room temperature. Followed by washed with TBST, the membranes were incubated with secondary antibody conjugated with Alexa Fluor® Plus 488 goat-anti-mouse IgG (1 : 1000; #A32723, RRID : AB_2633275; Invitrogen, Shanghai, China) at room temperature for 1 h. GeneGnome 5 (Synoptics Ltd., UK) was used to observe the protein bands on the membranes after coloration via ECL chemiluminescence kit (Advansta, USA).
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