SDS-PAGE gels were blotted with Trans-Blot Turbo mini 0.2 µm PVDF membranes (Bio-rad) using the Trans-Blot Turbo transfer system (Bio-rad) according to the manufacturer’s instructions. After transfer, the membranes were blocked with 50 mL 5% bovine serum albumin in 1× TBS with 0.1% Tween-20 (TBST) for 1 h. The membranes were then washed 5 times with 1× TBST. Following washing, the membranes were incubated 1 mg L−1 of anti-6X-His tag monoclonal antibody [HIS.H8] with an HRP conjugate (ThermoFisher) suspended in 10 mL 1× TBST for 0.5 h, and washed 3 times to remove unbound antibody with 1× TBST. To activate the HRP conjugate, membranes were incubated with ECL substrate for western blotting (Bio-rad) according to the manufacturer’s instructions. Chemiluminescence was imaged with the Amersham Imager 600 (GE Healthcare) according to the manufacturer’s instructions. Raw image files can be found in the supplemental information. Two technical replicates of each experiment were performed for each condition. Proteins expressed and purified at different conditions confirm the reproducibility of the observed phosphorylation ratios.
Trans blot turbo mini 0.2 m pvdf membranes
Manufactured by Bio-Rad
The Trans-Blot Turbo mini 0.2 µm PVDF membranes are a type of laboratory equipment designed for protein transfer in western blotting applications. These membranes have a pore size of 0.2 micrometers and are made of polyvinylidene fluoride (PVDF) material.
Automatically generated - may contain errors
2 protocols using trans blot turbo mini 0.2 m pvdf membranes
1
Western Blot Validation of 6X-His Tag Proteins
2
Western Blot Analysis of 6xHis-Tagged Proteins
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SDS-PAGE gels were blotted with Trans-Blot Turbo mini 0.2µm PVDF membranes (Bio-rad) using the Trans-Blot Turbo transfer system (Bio-rad) according to the manufacturer's instructions. After transfer, the membranes were blocked with 50 mL 5% bovine serum albumin in 1X TBS with 0.1% Tween-20 (TBST) for 1 hour. The membranes were then washed 5 times with 1X TBST. Following washing, the membranes were incubated 1 mg L -1 of anti-6X-His tag monoclonal antibody [HIS.H8] with an HRP conjugate (ThermoFisher) suspended in 10 mL 1X TBST for 0.5 hours, and washed 3 times to remove unbound antibody with 1X TBST. To activate the HRP conjugate, membranes were incubated with ECL substrate for western blotting (Bio-rad) according to the manufacturer's instructions. Chemiluminescence was imaged with the Amersham Imager 600 (GE Healthcare) according to the manufacturer's instructions. Raw image files can be found in the supplemental information (ST6). Two technical replicates of each experiment were performed for each condition. Proteins expressed and purified at different conditions confirm the reproducibility of the observed phosphorylation ratios.
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