After adding 50 μl of SDS sample buffer, the samples were heated at 95°C for five minutes, and centrifuged for one minute at 13,000 × g.
After the addition of SDS-PAGE buffer the samples were heated to 95°C and analyzed by Western blot using an anti-HA antiserum.
After adding 0.2 ml 2 × SDS PAGE buffer, the samples were heated at 99°C for 10 min to lyse the cells and denature proteins.
20 μL of lysate was mixed with 10 μL of 3× SDS-PAGE gel loading buffer (NEB), samples were heated to 95°C for 5 minutes and loaded on 10-20% polyacrylamide/Tris-glycine gel (Novex), run at 150 V in 1× Laemmli buffer, electroblotted to Immobilon or Immobilon-FL PVDF membrane in 1× Towbin buffer.
The immunoprecipitate was then washed three times with lysis buffer, resuspended in 20 μl of SDS sample buffer, and the samples were heated for 5 min at 100°C.
SDS-Laemmli buffer was added and samples were heated at 65 °C for 5 min.
To each sample 3 μL loading buffer was added and samples were heated for 10 minutes at 90 °C.
25 μl of SDS sample buffer was added, the samples were heated for 3 min at 95°C and proteins were separated on 7.5% SDS PAGE.
An equal volume of standard 2× Laemmli Sample Buffer was added and samples were heated at 95°C for 5 minutes and then centrifuged at 14,000 rpm for 5 minutes at 4°C.
