Cell and tissue extracts were collected in radioimmunoprecipitation (RIPA) lysis buffer and protein concentrations were measured by Bradford assay (Bio-Rad) to equalize loading. Samples were boiled for 5 minutes in SDS buffer before being analyzed on polyacrylamide gels, transferred to nitrocellulose membrane (0.2 micron) and blocked for one hour in 5% milk 0.1% tween-20 tris buffered saline (TBST). All membranes were incubated overnight with primary antibody (GAPDH – Abcam # Ab8245; Stathmin-2 – Novus # NBP1-49461) immunoblots were then washed three times with TBST and probed with horseradish peroxidase conjugated secondary antibodies diluted 1:5,000–1:10,000 in 5% milk for 1 hour at room temperature before being exposed to films or imaged on a LICOR Fc imager.
Ab8245
Manufactured by Novus Biologicals
Ab8245 is a recombinant monoclonal antibody produced in mouse. It is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Nbp1 49461, by Novus Biologicals (1 mentions)
Fc imager, by LI COR (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Nbp2 37740, by Abcam (1 mentions)
Supersignal west pico trial kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
2 protocols using ab8245
1
Western Blot Protein Detection Protocol
2
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Briefly, collected AF cells were lysed on ice in RIPA buffer (Beyotime, China) for 15 minutes and then centrifuged at 15,000 × g for 15 minutes at 4°C to collect cell lysates. After measuring protein concentration using a BCA Protein Assay Kit (Beyotime, China), equal protein samples in each group were resolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). After the PVDF membranes were incubated with primary antibodies (GAPDH: Abcam, ab8245; p16: Novus, NBP2-37740; p53: Abcam, ab26) overnight, they were incubated with secondary antibodies for 2 hours at room temperature. Finally, protein bands were visualized using the SuperSignal West Pico Trial Kit (Thermo, USA). Densitometric analysis of the protein bands was finished using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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