WB
Western blot analysis of Phospho-eIF4EBP1 (S65) on different lysates with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody at 1/1,000 dilution.
Lysates/proteins at 20 µg/Lane.
Predicted band size: 13 kDa
Observed band size: 20 kDa
Exposure time: 3 minutes;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/1,000 dilution was used in primary antibody dilution at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of 293T cells starved for 24 hours then treated with 100nM insulin for 30 minutes labeling Phospho-eIF4EBP1 (S65) with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody at 1/1,000 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.
IP
Phospho-eIF4EBP1 (S65) was immunoprecipitated from 0.2 mg 293T cell lysate with Phospho-eIF4EBP1 (S65) Recombinant Rabbit Monoclonal Antibody at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using Phospho-eIF4EBP1 (S65) Recombinant Rabbit Monoclonal Antibody at 1/1,000 dilution. Alpaca anti-Rabbit IgG Fc secondary antibody at 1/50,000 dilution was used for 1 hour at room temperature.
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 18 seconds;