The cell samples were lysed by cell lysate buffer (Beyotime, P0013) and later centrifuged to collect the supernatant, which was boiled for ten minutes to obtain the protein samples, which were then electrophoresed and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, ISEQ000010). The transfer membranes were blocked in 5% non-fat milk at room temperature for 1 h, and then incubated with the primary antibodies of YTHDF1 (Proteintech, #17479-1-AP), PKM2 (Abcam, #ab85542), HIF1α (ABclonal, #A17906), HIF2α (Abcam, #ab109616) and β-actin (ABclonal, #AC004) at 4 °C overnight, following the standard procedures from manufacturer’s instructions. Afterward the samples were incubated with secondary antibodies and washed three times. Finally, the electrochemiluminescence (Solarbio, # PE0010) signal was captured on the gel imaging system (BIO-RAD).
Cell lysate buffer
Manufactured by Beyotime
Sourced in China
Cell lysate buffer is a solution used to extract and solubilize cellular contents, including proteins, from biological samples. It is designed to disrupt cell membranes and release the contents of the cells without denaturing or altering the structure of the extracted components.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ac004, by ABclonal (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Ab233386, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Anti pgk1, by Abcam (1 mentions)
β actin, by ABclonal (1 mentions)
5 protocols using cell lysate buffer
1
Protein Expression Analysis by Western Blot
2
Protein Expression Analysis in SK-HEP-1 and MCF-7 Cells
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When SK-HEP-1 and MCF-7 cells grew to a density of about 80%, we collected them and added cell lysate buffer (Beyotime, Shanghai, China) and isolated the protein. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Boster, Wuhan, China). After SDS–PAGE electrophoresis, proteins were transferred to PVDF membrane. The membrane was incubated with the anti-S1PR1 antibody (Abcam, ab233386) for one night and then an anti-rabbit secondary antibody (Sanjian, Tianjin, China, LK2001) for 2 h. Images were processed with Image J software.
3
Bax and Bcl2 protein levels in CHF
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Peripheral blood samples were drawn from CHF patients at admission, followed by centrifugation (1000 × g, room temperature, 10 minutes) to separate blood cells and serum. We used density gradient centrifugation to analyze peripheral blood mononuclear cells (PBMC), and added cell lysate buffer (P0013C, Beyotime) after cell counts to release Bax and Bcl2 proteins. And then we used human Bcl2 associated X protein (Bax) ELISA Kit (EK12824, Signalway Antibody) to detect levels of Bax protein in lysis of PBMC, and used human B-Cell leukemia/lymphoma 2 (Bcl2) ELISA Kit (EK12835, Signalway Antibody) to detect levels of Bcl2 protein in lysis of PBMC.
4
Quantification of Cellular GSH
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H9C2 cells were treated as described above, and then cell lysates were prepared using a cell lysate buffer (Beyotime). The GSH levels in the cell lysates were determined using the GSH assay kit, following the manufacturer's protocol. Briefly, GSH was reacted with 5,5′-dithiobis (2-nitrobenzoic acid) to produce GSSG and 5′-thio-2-nitrobenzoic acid (TNB). The TNB absorbance was measured at 405nm, and the GSH content was calculated based on the TNB concentration. The final GSH content was expressed as micromoles per gram protein.
5
Protein Expression Analysis by Western Blot
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The cell samples were washed the cells with PBS and lysed with cell lysate buffer (Beyotime Biotechnology, cat. #P0013) and then centrifuged (13,500 g, 4°C for 15 min) to collect the supernatant.
Protein sample was transferred to polyvinylidene uoride (PVDF) membrane (Millipore, Billerica, MA, USA, cat. #ISEQ-000010). The transfer PDVF membrane was blocked in 5% non-fat milk for 1 hour at room temperature, and then incubated with primary anti-YTHDF3 antibodies (Proteintech, Cat No: 25537-1-AP, 1:2000), anti-PGK1 (Abcam, Cat No: ab199438, 1:2000) and β-actin (ABclonal, Cat No. #AC004). Protein sample was separated on a polyacrylamide gel at 4°C overnight according to manufacturer's instructions.
After incubation with secondary antibody, electrochemiluminescence (Solarbio, Beijing # PE-0010) signal was captured on the Bio-Rad gel imaging system.
Protein sample was transferred to polyvinylidene uoride (PVDF) membrane (Millipore, Billerica, MA, USA, cat. #ISEQ-000010). The transfer PDVF membrane was blocked in 5% non-fat milk for 1 hour at room temperature, and then incubated with primary anti-YTHDF3 antibodies (Proteintech, Cat No: 25537-1-AP, 1:2000), anti-PGK1 (Abcam, Cat No: ab199438, 1:2000) and β-actin (ABclonal, Cat No. #AC004). Protein sample was separated on a polyacrylamide gel at 4°C overnight according to manufacturer's instructions.
After incubation with secondary antibody, electrochemiluminescence (Solarbio, Beijing # PE-0010) signal was captured on the Bio-Rad gel imaging system.
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