Cells were lysed in the lysis buffer (20 mM HEPES pH7.4, 150 mM NaCl, 1% NP-40, 2 mM EDTA and a phosphotase inhibitor cocktail purchased from Roche (Basel, Switzerland). Two micro grams of total protein was mixed with the loading buffer without SDS and β-ME. The separating gel and stacking gel were made without SDS. The running buffer was in pH 8.3 without SDS. The gel was powered up at a current of 20 to 25 mA constant current. The gel was run until the bromophenol blue dye front reached the bottom. Then, samples were transferred to PVDF membrane for 120 min at 300 mA. After transfer, the membrane was incubated in 20 mL of 8% acetic acid for 15 min to fix the protein. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Then, the membrane incubated with the primary antibodies in blocking buffer at 4 °C overnight before detection with HRP-conjugated secondary antibodies. Chemiluminescence was detected with ECL solution. Every test was repeated at least twice.
Phosphotase inhibitor cocktail
Manufactured by Roche
The Phosphotase inhibitor cocktail is a laboratory reagent designed to inhibit the activity of phosphatase enzymes. Phosphatases are responsible for the removal of phosphate groups from proteins, and their inhibition can be useful in various biochemical and cell-based assays.
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Lab products found in correlation
P62 sqstm1, by Abnova (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Gapdh, by OriGene (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
2 protocols using phosphotase inhibitor cocktail
1
Western Blot Protocol for Protein Analysis
2
Western Blot Analysis of Autophagy Markers
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Cells were lysed in RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Roche) and phosphotase inhibitor cocktail (Roche). Sample lysates were boiled with Laemmli sample buffer and loaded in gel for electrophoresis. After blot transfer, polyvinylidene difluoride membranes were blocked with blocking buffer (LI-COR) and incubated with primary antibodies overnight. After 3× washing with PBS, secondary antibodies were applied for 1 h and then washed with TBST (0.05% Tween 20) before imaging. Blots were scanned by the Odyssey imager (LI-COR), and integrated intensities using the top and bottom of the bands as background were quantified by the Odyssey software. Primary antibodies such as LC3 (Novus), p-ERK (Cell Signaling), p-AKT (Cell Signaling), p62 (SQSTM1) (Abnova), and GAPDH (Origene) were used for this experiment.
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