Total protein was extracted from the cells using 1 × RIPA lysis buffer (P10013B, Beyotime, Shanghai, China). The concentration of protein was measured by using BCA Protein Assay Kit (P0010, Beyotime, Shanghai, China). And then equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membrane (NC membrane). After electroblotting, the membranes were blocked with PBS-5% fat-free dried milk at room temperature for 1 h and incubated with primary antibodies for LDHA (1:2000, Santa Cruz sc-137243, Texas, USA), PFKM (1:1000, ABclonal A5477, Shanghai, China), NAT10 (1:1000, ABclonal A19286, Wuhan, China), YTHDC1 (1:500, Proteintech 14,392–1-AP, Wuhan, China), ac4C (1:1000, Abcam ab253039, Cambridge, UK) or β-actin (1:5000, Abcam ab253039, Cambridge, UK). The membrane was then incubated with anti-mouse secondary antibodies (RS23910, ImmunoWay, USA) or anti-rabbit secondary antibodies (RS23920, ImmunoWay, USA). Results were detected using Odessey Clex (LI-COR, USA), followed by further analysis.
Odessey clex
Manufactured by LI COR
Sourced in United States
The Odyssey CLx is a near-infrared imaging system designed for quantitative Western blot analysis and in-gel fluorescence detection. It utilizes two-color fluorescence detection technology to provide simultaneous quantitation of target proteins and loading controls in a single blot or gel.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using odessey clex
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of Protein Targets
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Cell samples were washed using PBS, and then the total proteins were extracted using 1×RIPA lysis buffer (Beyotime, Shanghai, China). The lysates were fully crushed by ultrasonic and cleared by high-speed centrifugation at 13,500g for 15 min. The extracted total proteins were quanti ed by BCA Protein Assay Kit (Beyotime, Shanghai, China), then equal amounts of proteins were separated by SDS-PAGE and transferred to NC membrane. After the membranes were blocked with 5% w/v non-fat milk for 1h, the proteins were probed at 4℃ overnight using different primary antibodies METTL1 (14994-1-AP, proteintech), FTH1 (#4393, CST), GPX4 (A13309, Abclonal), GAPDH (AC002, Abclonal), β-Tubulin (AC021, Abclonal). On the next day, the membranes were incubated with a secondary antibody (RS23910, ImmunoWay) for 1 h at room temperature. Results were detected using Odessey Clex (LI-COR, America), followed by further analysis.
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