Proteins were extracted 48 h after the transfection. Protein samples were resolved by electrophoresis in a 12% SDS-PAGE gel (Genscript; M00667) and were transferred to a polyvinylidene difluoride membrane in transfer buffer (Genscript; M00139) using a wet procedure at 300 mA for 1 h. Followed by blocking with 5% nonfat milk dissolved in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% (v/v) Tween 20) at room temperature (RT) for 1 h, the membranes were probed with anti–Flag-Tag mouse monoclonal antibody (1:2000, CWBIO, CW0287) and anti–β-tubulin mouse monoclonal antibody (1:2000, CWBIO, CW0098) overnight at 4 °C. The membrane was washed for 10 min with TBST buffer on an orbital shaker for three times and then incubated with goat antimouse secondary antibody (1:2000, CWBIO, CW0102S) at RT for 1 h and washed with TBST as described previously. Proteins were detected with ECL Western horseradish peroxidase substrate (Advansta) on a chemiluminescent gel imaging system (MicroChemi, DNR). EasySee Western Marker (Transgen; DM201-01) was used as molecular weight marker.
Easysee western marker
Manufactured by Transgene
Sourced in China
EasySee Western Marker is a pre-stained protein molecular weight marker for Western blotting. It provides consistent and reliable molecular weight determination of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Cw0102, by CWBIO (1 mentions)
Cw0098, by CWBIO (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Ac033, by Beyotime (1 mentions)
A00227, by Boster Bio (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ba0412, by Boster Bio (1 mentions)
Ba0315 2, by Boster Bio (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
3 protocols using easysee western marker
1
Western Blot Analysis of Flag-Tagged Proteins
2
Western Blot Analysis of Protein Expression
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Cells cultured in 60-mm dishes were lysed with RIPA Lysis Buffer (Beyotime, Haimen, China) supplemented with protease inhibitor cocktail (Sigma). Equal protein content of total cell lysates were mixed in loading buffer, boiled for 5 min, and then subjected to SDS-PAGE gel (10%) electrophoresis along with EasySee western marker (TransGen Biotech, Beijing, China). After electrophoresis, proteins were blotted onto nitrocellulose membranes and blocked with 5% bovine serum albumin, probed with a primary antibody at 4 °C overnight, followed by a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then enhanced chemiluminesence detection (Amersham Bioscience, Piscataway, NJ).
3
Western Blot Analysis of Apoptosis-Related Proteins
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RIPA Lysis Buffer (Beyotime) was used to extract total protein, and BCA Protein Assay Kit (Beyotime) was used to quantify the protein. Afterwards, protein samples (30 µg) were separated by 10% SDS-PAGE gel and transferred to PVDF membranes (Beyotime). Next, the membranes were blocked with skimmed milk for 2 h. After incubating with primary antibodies against Bcl-2 (26Kda, 1:2,000, BA0412, Boster, Wuhan, China), Bax (20Kda, 1:1,500, BA0315-2, Boster), Cleaved-caspase 3 (17Kda, 1:1,000, AC033, Beyotime), FOXO1 (82Kda, 1:2,000, AF603, Beyotime), or GAPDH (36Kda, 1:2,000, A00227, Boster), the membrane was then incubated with HRP Conjugated AffiniPure Goat Anti-rabbit/mouse IgG (H + L) (1:10,000, BA1056, Boster). The protein signals were visualized using BeyoECL Star (Beyotime). EasySee Western Marker (25-90Kda, DM201-01, Transgen Biotech, Beijing, China) was used as a molecular weight standard.
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