Total protein extracts were isolated in lysis buffer as previously described [22 (link)]. Equal amounts of proteins (10–20 μg) were resolved using SDS-PAGE gels and transferred to PVDF Hybond-p membrane (GE Healthcare). Membranes were blocked with I-block (Life Technologies) for at least 2 hours, under rotation at RT. Membranes were then incubated overnight at 4°C under constant shaking with the following primary antibodies: HIF-1α (mouse, 1 : 250, BD Pharmingen), mTOR total, mTOR (S2448), P70S6K total, P70S6K (T389), AKT total, AKT (T308), AKT (S473), BAX, PARP, (all rabbit, 1 : 1000, Cell Signaling Technologies), and β-actin (mouse, 1 : 10000, Sigma Aldrich) as loading control. Membranes were next incubated with peroxidase-labeled goat anti-rabbit IgG or goat anti-murine IgG (both 1 : 50.000 in I-block from Sigma Aldrich) for 60 min. All membranes were visualized using ECL Select (GE Healthcare) and exposed to Hyperfilm MP (GE Healthcare).
I block
Manufactured by Merck Group
The I-block is a laboratory equipment designed for the containment and manipulation of samples or materials. It provides a controlled environment for various experimental and analytical processes. The core function of the I-block is to isolate and maintain the integrity of the samples or materials during handling and testing.
Automatically generated - may contain errors
Lab products found in correlation
I block, by Thermo Fisher Scientific (2 mentions)
Hybond, by Cytiva (2 mentions)
Hybond p pvdf membrane, by GE Healthcare (1 mentions)
Hif 1α, by BD (1 mentions)
Hyperfilm mp, by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Goat anti rabbit igg, by Merck Group (1 mentions)
Ecl select, by GE Healthcare (1 mentions)
Rabbit anti flag, by Merck Group (1 mentions)
Goat anti rabbit igg h l, by Abcam (1 mentions)
Hybond ecl membrane, by GE Healthcare (1 mentions)
G box, by Syngene (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
2 protocols using i block
1
Western Blot Analysis of Protein Targets
2
Immunoblotting of Pull-Down Assay Samples
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Cell lysates or supernatants from the pull-down assay were mixed with 4× Laemmli buffer (to the final concentration of 1× Laemmli buffer), incubated at 85°C for 10 min and separated on a 10% polyacrylamide gel in SDS running buffer (190 mM glycine, 0.1% SDS, 25 mM Tris, pH 8.3). Proteins were transferred to Hybond ECL membranes (GE Healthcare) in transfer buffer (190 mM glycine, 20% methanol, 25 mM Tris, pH 8.3) at 350 mA for 2 h. Membranes were washed with deionized water and blocked overnight at 4°C in a solution of 0.2% I-Block (Life Technologies) in PBS with 0.1% Tween-20 (PBS-T). Blocked membranes were incubated for 1.5 h with a solution of primary antibodies (rabbit anti-FLAG (Sigma) in 0.2% I-Block, washed four times with PBS-T, incubated with a solution of HRP conjugated secondary antibodies (Goat Anti-Rabbit IgG H&L (Abcam)) and again washed four times with PBS-T. Detection was performed with SuperSignal West Pico Chemiluminescent Substrate (Life Technologies) in the G:BOX (Syngene) detector and the figures processed using the ImageJ software.
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