Rabbit anti β actin conjugated to alexafluor680

Cell lines were harvested during active growth (0.4E6 – 0.6E6 cells/mL) and lysed in RIPA buffer supplemented with protease inhibitor cocktail (Halt). Lysates were centrifuged at 16,000 xg for 20 min at 4 °C, and total protein concentration was quantified by Pierce BCA Assay (Thermo). SDS loading buffer was added to 50 μg total protein, and samples were boiled at 95 °C for 5 min. Samples were loaded on 4–15% pre-cast PAGE gels (Bio-Rad), run at 85 V in running buffer, and transferred to a PVDF membrane (Bio-Rad). Membranes were incubated for 1 hour in blocking buffer (1% (w/v) BSA in 0.1% TBS-T), followed by an overnight incubation at 4 °C in blocking buffer containing primary antibody. Membranes were washed for 10 min 0.1% TBS-T three times, followed by a 1 hr incubation with secondary antibody with rotation at room temperature and protected from light. Membranes were washed for 10 min 0.1% TBS-T three times and imaged using an LI-COR Odyssey DLx.
Antibodies used for Western blotting include rabbit anti-IGHMBP2 at 1:500 (Proteintech, 23945–1-AP), rabbit anti-β-actin conjugated to AlexaFluor680 (Abcam); rabbit anti-eIF2α at 1:1,000; rabbit anti-eIF2α phospho-S51 at 1:1,000. Membranes were incubated with goat anti-rabbit 800 CW secondary antibody (LI-COR, 926–32211) at 1:10,000.

Cell lines were harvested during active growth (0.4 × 10⁶–0.6 × 10⁶ cells/ml) and lysed in RIPA buffer supplemented with protease inhibitor cocktail (Halt). Lysates were centrifuged at 16,000g for 20 min at 4°C, and total protein concentration was quantified by Pierce BCA Assay (Thermo Fisher Scientific). SDS loading buffer was added to 50 μg total protein, and samples were boiled at 95°C for 5 min. Samples were loaded on 4–15% pre-cast PAGE gels (Bio-Rad), run at 85 V in running buffer, and transferred to a PVDF membrane (Bio-Rad). Membranes were incubated for 1 h in blocking buffer (1% [wt/vol] BSA in 0.1% TBS-T), followed by an overnight incubation at 4°C in blocking buffer containing primary antibody. Membranes were washed for 10 min with 0.1% TBS-T three times, followed by 1 h incubation with secondary antibody with rotation at RT and protected from light. Membranes were washed for 10 min 0.1% TBS-T three times and imaged using a LI-COR Odyssey DLx.
Antibodies used for Western blotting include rabbit anti-IGHMBP2 at 1:500 (23945-1-AP; Proteintech), rabbit anti-β-actin conjugated to Alexa Fluor 680 (Abcam); rabbit anti-eIF2α at 1:1,000; rabbit anti-eIF2α phospho-S51 at 1:1,000. Membranes were incubated with goat anti-rabbit 800 CW secondary antibody (926-32211; LI-COR) at 1:10,000.