First, isolated mRNA (200 ng for each group) was denatured by heating at 95 °C for 3 min, followed by chilling on ice immediately. Dilutions were spotted on an Amersham Hybond-N + membrane optimized for nucleic acid transfer (GE Healthcare). The membrane was crosslinked under UV light and washed with 1× PBST buffer. After the membrane was blocked with 5% nonfat milk in PBST, it was incubated with anti-m6A antibody (202003, Synaptic Systems) overnight at 4 °C. Then, the membrane was incubated with secondary antibody at room temperature for 1 h. Immunoblots were developed using a chemiluminescent reagent (Beyotime). The same 200 ng of mRNAs were spotted on the membrane, stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) for 2 h and washed with RNase-free water for 1 h.
Chemiluminescent reagent
Manufactured by Beyotime
Sourced in China
Chemiluminescent reagents are a class of laboratory chemicals used in various analytical and experimental techniques. They produce light through a chemical reaction, which can be detected and measured. These reagents are commonly used in applications such as immunoassays, reporter gene assays, and Western blotting to quantify and analyze specific target molecules or proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Amersham hybond n membrane, by GE Healthcare (1 mentions)
Anti m6a antibody 202 003, by Synaptic Systems (1 mentions)
Hybond, by Cytiva (1 mentions)
Df6894, by Affinity Biosciences (1 mentions)
Ab188570, by Abcam (1 mentions)
Df6087, by Affinity Biosciences (1 mentions)
Af5103, by Affinity Biosciences (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
Df7561, by Affinity Biosciences (1 mentions)
Ap0489, by ABclonal (1 mentions)
Af5142, by Affinity Biosciences (1 mentions)
Af7018, by Affinity Biosciences (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Mouse anti gapdh, by Proteintech (1 mentions)
Hrp conjugated secondary antibody, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Mouse anti gabab1, by Abcam (1 mentions)
9 protocols using chemiluminescent reagent
1
Quantifying mRNA m6A Modifications
2
Mechanotransduction and Anti-inflammatory Effects
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Western blotting was performed to assess the influence of the
CAV1-YAP-mediated mechanotransduction pathway and the anti-inflammatory
effects of the NPC-encapsulated composite hydrogel under inflammatory
conditions. Proteins were labeled and incubated overnight at 4°C with the
following primary antibodies: YAP (1:500, A1001, ABclonal), p-YAP (1:500,
AP0489, ABclonal), CAV1 (1:2000, 16447-1-AP, Proteintech), Col II (1:2000,
ab188570, Abcam), Agg (1:2000, DF7561, Affinity), IL-1β (1:1000, AF5103,
Affinity), IL-6 (1:1000, DF6087, Affinity), IL-10 (1:1000, DF6894,
Affinity), IL-4 (1:1000, AF5142, Affinity), and β-actin (1:5000, AF7018,
Affinity). After washing using TBST buffer, the membranes were treated with
horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, S0001,
Affinity) for 2 h at room temperature. Then examined with a chemiluminescent
reagent (Beyotime), and quantified the signal intensity using ImageJ
software.
CAV1-YAP-mediated mechanotransduction pathway and the anti-inflammatory
effects of the NPC-encapsulated composite hydrogel under inflammatory
conditions. Proteins were labeled and incubated overnight at 4°C with the
following primary antibodies: YAP (1:500, A1001, ABclonal), p-YAP (1:500,
AP0489, ABclonal), CAV1 (1:2000, 16447-1-AP, Proteintech), Col II (1:2000,
ab188570, Abcam), Agg (1:2000, DF7561, Affinity), IL-1β (1:1000, AF5103,
Affinity), IL-6 (1:1000, DF6087, Affinity), IL-10 (1:1000, DF6894,
Affinity), IL-4 (1:1000, AF5142, Affinity), and β-actin (1:5000, AF7018,
Affinity). After washing using TBST buffer, the membranes were treated with
horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, S0001,
Affinity) for 2 h at room temperature. Then examined with a chemiluminescent
reagent (Beyotime), and quantified the signal intensity using ImageJ
software.
3
Western Blot Protein Analysis Protocol
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Total protein was extracted using RIPA lysis buffer with protease inhibitor cocktail. The protein concentrations were normalized with a BCA assay kit (Thermo Fisher, USA). Proteins from each group were loaded onto SDS-PAGE gels and separated before they were transferred to PVDF membranes (Millipore). The membrane was incubated with primary antibody against each target protein at 4 °C overnight. Afterward, the membrane was incubated with secondary antibody at room temperature for 1 h. Immunoblots were developed using a chemiluminescent reagent (Beyotime, China) according to the manufacturer’s instructions73 (link). The antibodies used are listed in Supplementary Table 6 .
4
TRPV1 and GABAB1 Expression in CFA-Induced Neuroinflammation
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SD rats were divided into the control and CFA groups, plantar injection three days later, and fresh brain tissues containing the CSF-contacting nucleus were collected. Ten times the tissue weight of RIPA lysis buffer (Beyotime, China) and 1/100 of the lysate volume of PMSF (Beyotime, China) was added. After the tissues were homogenized and centrifuged at 4°C for 15 min at 12,000 rpm, the supernatant was collected, and the total protein concentration of each sample was determined by a BCA protein assay kit (Beyotime, China). Then, the proteins were separated by 8% SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membranes, which were blocked with 5% skim milk powder for 2 h and incubated with rabbit anti-TRPV1 (1:2000, Novus Biologicals), mouse anti-GABAB1 (1:500, Abcam) and mouse anti-GAPDH (1:2000, Proteintech) antibodies at 4°C overnight. After half an hour at room temperature, the PVDF membranes were rinsed three times with TBST (TBS containing 1% Tween-20) and incubated with corresponding HRP-conjugated secondary antibodies (1:2000, Beyotime, China) for 2 h. Then, the membranes were rinsed six times with TBST. The protein bands were detected by a chemiluminescent reagent (Beyotime, China), and the relative grayscale values were analyzed using ImageJ software.
5
VDAC1 Knockdown Protein Expression Analysis
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72 hours after transfection of si-VDAC1, A549 cells were collected and incubated in 200 μ L 1∗SDS-PAGE Sample Loading Buffer (Cat No. P0015L), 98°C for 10 minutes until the cells were completely lysis. Proteins were resolved on 10% gel using PAGE gel quick preparation kit (10%, CAT: PG112) and transferred to Trans-blot Turbo nitrocellulose membranes (Bio-Rad). Then, incubate the membrane in 10% milk and seal it for 2 hours. For primary antibody–protein hybridization, membranes were probed with the following antibodies at 4°C overnight (Beyotime): LDHA Rabbit Polyclonal Antibody, CAT: AF0216; COX IV Rabbit Polyclonal Antibody, CAT: AF6549; Hexokinase II Rabbit Polyclonal Antibody, CAT: AF7080; SDHB Rabbit Polyclonal Antibody, CAT: AF7956; and PGK1 Rabbit Monoclonal Antibody, CAT: AF1825. Thereafter, secondary anti-mouse (Beyotime CAT: LF101) or anti-rabbit IgG antibodies (Beyotime CAT: LF102) were incubated for 1 h at room temperature. Protein bands were developed with chemiluminescent reagents (Beyotime) and imaged with a Tanon 5200.
6
Protein Extraction and Western Blot Analysis
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T24 and 5637 cells were treated with RIPA reagents (Beyotime), and then the total protein was obtained by centrifugation (12,000 × rpm, 10 min). The protein was quantified with BCA protein assay kit (beyotime). 50 μg protein/lane was isolated in 10% SDS-PAGE, and then transferred to PVDF membrane. The membranes were sealed for 2 h at room temperature with 5% skimmed milk, and then incubated with primary antibody [xCT (1:1000, ab175186, Abcam); GPX4 (1:1000, ab40993, Abcam); Bcl-2 (1:1000, ab117115, Abcam); Bax (1:1000, ab3191, Abcam); Cytochrome C (1:1000, ab13354, Abcam); PI3K (1:1000, #4292, Cell Signaling Technology); p-PI3K p85α (1:1000, #17,366, Cell Signaling Technology); AKT (1:1000, #9272, Cell Signaling Technology); p-AKT Thr308 (1:1000, #13,038, Cell Signaling Technology); p-AKT Ser473 (1:1000, #4060, Cell Signaling Technology); mTOR (1:1000, #2972, Cell Signaling Technology); p-mTOR Ser2448 (1:1000, #5536, Cell Signaling Technology); β-actin (1:1000, ab8224, Abcam)] overnight at 4°C. Then the membranes were incubated for 2 h at room temperature with second antibody [Goat Anti-Rabbit IgG H&L (1:5000, ab96899, Abcam) or Goat Anti-Mouse IgG H&L (1:5000, ab96879, Abcam)]. The bands were visualized with chemiluminescent reagents (Beyotime) and photographed on a gel image analysis system (Bio-Rad, USA).
7
Protein Expression Analysis of Cell Spheroids
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Cell spheroids were subjected to low-speed centrifugation to separate the culture medium and washed thrice with DPBS. Proteins were extracted using RIPA buffer supplemented with Phosphatase and Protease Inhibitor Cocktails (Beyotime) and quantified utilizing a BCA kit (Solarbio). Proteins were resolved on a 12% SDS polyacrylamide gel and transferred onto a PVDF membrane (Millipore, MO, USA) using a wet transfer process. The membrane was then incubated with primary antibodies against β-actin, CD31, VEGFA, RUNX2, ALP, and OCN overnight at 4°C. The following day, the membrane was exposed to an HRP-conjugated secondary antibody, imaged using chemiluminescent reagents (Beyotime).
8
Western Blot Analysis of Vascular Proteins
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The protein extracts were extracted from mice blood vessels and HAECs. Proteins were separated in sodium dodecyl sulfate-polyacrylamide electrophoresis gels (8–15%) and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The PVDF membranes containing the target protein was sealed with 5% skimmed milk for about 2 h. Then, incubate the first antibodies and stay overnight at 4 °C. After the PVDF membranes were cleaned with TTBS, the second antibodies were incubated. After the above operations are completed, PVDF membranes were in full contact with chemiluminescent reagents (Beyotime). The final images were captured by a Bio-Rad imaging system and analyzed with a Gel-Pro Analyzer Version 4.0 (Media Cybernetics, MD, USA).
9
Quantitative Proteome Analysis of NRVM Cells
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Total proteins were extracted from NRVM cells. NRVM cells lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffers (Beyotime, Nanjing, China). Cell lysates were centrifuged at 12,000 × g for 15 min. The proteins concentration was detected by the BCA method, and equal quantities of protein extracts were loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Bedford, MA, U.S.A.). The membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4°C. After washing 3 times with TBST, the membranes were incubated with the HRP-linked secondary antibody at room temperature for 1 h, and then washed 3 times with TBST again. Finally, the protein bands on the membranes were detected by chemiluminescent reagents (Beyotime, Nanjing, China). Chemiluminescence signals were quantified using an ECL imager, and analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA). The specific primary antibodies were: anti-GAPDH (Proteintech), anti-Histone3 (Proteintech), anti-H3K4me3 (CST), anti-H3K9me3 (CST), anti-H3K27me3 (CST), anti-H3K36me3 (CST), anti-Flag (Sigma), and anti-Puromycin (Santa Cruz).
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