Fractions added with SDS-PAGE loading buffer were boiled at 100 °C for 10 min, then centrifuged (4 °C, 12,000g, 10 min) before loading on gels (TGX Stain-Free™ FastCast™ Acrylamide Kit, 12%, Bio-Rad, Cat. No. 161-0185) for SDS-PAGE.
Equal amounts (75 µg) of cytoplasmic or nuclear homogenate in 4× Laemlli buffer were boiled at 95°C for 5 minutes then loaded in the wells of at 7.5% gel.
The washed immunoprecipitates, resuspended in Laemmli buffer, was boiled at 95 °C for 5 min before immunodetection of SAG, Bax, SARM, Bcl-2 or ubiquitin.
30 μg of cell lysates (1 % Nonidet buffer) were boiled for 10 min at 60 °C in Laemmli sample buffer.
The Transfection cell lysates/platelet cell lysates in 1X Laemelli buffer were boiled for 5', clarified by centrifugation at 14 000 RPM for 5', loaded onto a 6% SDS PAGE, and transferred onto immobilon nylon membrane.
Following protein determination (n=3, Bradford, 1976) and addition of sample buffer, samples were boiled at 95°C for 5 min and solubilised proteins (50 μg) were separated by sodium dodecyl sulphate (SDS) polyacrylamide gel (10%) electrophoresis.
Samples containing 20 μg of protein and 1× Laemmli buffer (Laemmli, 1970) were boiled at 95°C for 4 5 min.
After adding 5× loading buffer, protein samples were boiled at 95°C for 5 min for denaturation, after which the protein was separated using SDS-PAGE and then transferred onto a PVDF membrane.
Otherwise, the reactions were stopped with the addition of Laemmli buffer and the samples were boiled at 98°C for five minutes.
The other fractions were equally mixed with Laemmli buffer, and all samples were boiled at 95°C for 5 min and separated on a 4 12% gradient gel (Novex, Invitrogen).
After electrophoresis loading buffer was added, all samples were boiled at 100 °C for 5 min and then centrifuged at 13,000×g for 10 min, and 10 μL of each loaded onto a SDS-PAGE gel (TGX Stain-Free™ FastCast™ Acrylamide Kit, 12%, Bio-Rad, Cat. No. 161-0185).
