DEFs were transfected with pCAGGS-3D-HA expressing the 3D protein. Cells were lysed in 200 μl cell lysis buffer (Beyotime) containing 1% PMSF. The cell lysate was centrifuged, and the supernatant was collected. Samples were fractionated by SDS-PAGE electrophoresis and then transferred to PVDF membrane, blocked with 5% non-fat dry milk at room temperature for 5–6 h. The membranes were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. The membranes were washed three times with TBS-Tween and incubated for 1 h at 37 °C with the respective secondary antibodies diluted in blocking buffer. The membranes were then washed three times with TBS-Tween, and bound proteins were detected using an ECL chromogenic kit (Beyotime).
Ecl chromogenic kit
Manufactured by Beyotime
Sourced in China
The ECL chromogenic kit is a laboratory equipment designed for the detection and visualization of proteins using chemiluminescence. It provides a sensitive and reliable method for protein analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Apexbio (2 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Ab126605, by Abcam (1 mentions)
Ab286164, by Abcam (1 mentions)
Ab69325, by Abcam (1 mentions)
Hrp 60004, by Proteintech (1 mentions)
Bicinchoninic acid kit, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
6 protocols using ecl chromogenic kit
1
Western Blot Analysis of 3D Protein
2
Protein Quantification and Western Blot Analysis
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Proteins were first extracted using the radioimmunoprecipitation assay lysis solution with phenylmethylsulfonyl fluoride and protease inhibitor cocktail (APEXBIO, USA) and then measured using a bicinchoninic acid kit (Beyotime, China). SDS-polyacrylamide gel electrophoresis was used to separate the protein lysates, which were then transferred to polyvinylidene fluoride membranes. After blocking the membranes with 5% skim milk, they were incubated with specific primary antibodies overnight at 4 °C, followed by a 1-h incubation with secondary antibodies at room temperature. On the Mini-REPORT Tetra Electrophoresis System, proteins isolated on the membranes were visualized using an ECL chromogenic kit (Beyotime, China). The antibodies used in this study were as follows: FTO (Abcam; ab126605, 1:1,000), m6A (Abcam; ab286164, 1:1,000), METTL3 (Abcam; ab69325, 1:1,000), vimentin (CST; 5741T, 1:1,000), CDH1 (CST; 3195S, 1:1,000), Snail (Wanlei; WL01863, 1:1,000), IGF2BP2 (Proteintech; 11601-1-AP, 1:1,000), IGF2BP3 (Proteintech; 14642-1-AP, 1:1,000), DDIT4 (Proteintech; 10638-1-AP, 1:1,000), AKT (CST; 4685S, 1:1,000), TSC2 (Proteintech; 24601-1-AP, 1:1,000), p-AKT (CST; 13038S, 1:1,000), p-TSC-2 (Proteintech; 29000-1-AP, 1:1,000), AR (Proteintech; 22089-1-AP, 1:1,000), and GAPDH (Proteintech; HRP-60004, 1:1,000).
3
Western Blot Protein Detection Protocol
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Cells were lysed in 200 μl cell lysis buffer (Beyotime) containing 1% PMSF. The cell lysate was centrifuged, and the supernatant was collected. Samples were fractionated by SDS-PAGE electrophoresis and then transferred to PVDF membrane, which was blocked with 5% non-fat dry milk at room temperature for 5–6 h. The membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. The membranes were washed three times with TBS-Tween and incubated for 1 h at 37°C with the respective secondary antibodies diluted in blocking buffer. The membranes were then washed three times with TBS-Tween, and bound proteins were detected using an ECL chromogenic kit (Beyotime).
4
Western Blot Analysis of Protein Expression
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Total proteins were extracted from tissue or cell specimens using RIPA lysis buffer containing PMSF and Protease Inhibitor Cocktail (APEXBIO, USA) and protein concentrations determined using a BCA kit (Beyotime, China). Then, 30 μg of whole cell or tissue lysates were resolved on 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk /TBST, membranes were incubated with specific primary antibody at 4 °C overnight. Then, blots were washed with TBST, incubated with species-specific horseradish peroxidase (HRP) -conjugated secondary antibody (ABclonal, Cat.NO:AS014), and the signal visualized using an ECL chromogenic kit (Beyotime, China) on the Mini-REPORT Tetra Electrophoresis System. Antibodies used for western blot analysis were diluted as follows: METTL3 (Abcam, ab195352), EphA2 (CST, 6997), VEGFA (Proteintech,19003-1-AP), FTO (Abcam, ab126605), IGF2BP2 (Proteintech,11601-1-AP), IGF2BP3 (Proteintech,14642-1-AP), AKT/ p-AKT (CST, 1:1000), mTOR/p-mTOR (CST, 2983/5536), ERK1/2 and p-ERK1/2 (Proteintech, 16443-1-AP/80031-1-RR), HIF1α (CST, 36169), E-cadherin (CST, 3195), Vimentin (CST, 5741), and GAPDH (Abcam, ab181602). The original pictures of western blot are listed in the supplementary material.
5
m6A Dot Blot Quantification Protocol
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m6A dot blotting was performed as previously described [49 (link)]. Briefly, isolated total RNA was measured using a NanoDrop instrument, and 200–400 ng RNA was spotted onto a Hybond-N + membrane (Invitrogen, USA). Then, membranes were crosslinked using UV irradiation and incubated with m6A-specific antibody (Abcam, 1:1000 dilution) overnight at 4 °C, after blocking with 5% nonfat milk in PBST. Then, diluted HRP-conjugated secondary antibody (1:10000) was added to the membrane for 1 h at room temperature. Membranes were developed using an ECL chromogenic kit (Beyotime, China) and the signal detected using a the Mini-REPORT Tetra Electrophoresis System. Methylene blue staining was used as a loading control.
6
Western Blot Analysis of Protein Expression
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DEFs were cotransfected with the recombinant plasmid pRed-IRES-EGFP and DNAzymes. The cells were lysed in 100 μl of cell lysis buffer containing 1% PMSF (Beyotime). The cell lysate was centrifuged, and the supernatant was collected. Samples were fractionated by SDS–PAGE, transferred to PVDF membranes, and blocked with 5% nonfat dry milk at room temperature for 5–6 h. The membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. The membranes were washed three times with TBS-Tween and incubated for 1 h at 37°C with the respective secondary antibodies diluted in blocking buffer. The membranes were washed three times with TBS-Tween, and bound proteins were detected with an enhanced chemiluminescence (ECL) chromogenic kit (Beyotime).
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