Supernatants were subjected to electrophoresis using Any-KD Mini-Protean gels (Bio-Rad) and separated by SDS-PAGE. Gels were transferred onto nitrocellulose membrane (Bio-Rad) by semi-dry transfer (24 V, 0.5 A, 1 h). Membranes were blocked with Blocking One (Nacalai) for 30 minutes and incubated with primary antibodies ((anti-TGFPβ antibody (Abcam; clone 9016) anti-IL-1β antibody (Novus biological; NB600–633)) in Blocking One at 4°C. Membranes were washed in TBS-Tween20 (0.05%) and then incubated with HRP-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 hour at room temperature. Membranes were washed in TBS-T again before signal was developed using the SuperSignal West Femto Maximum Sensitivity Substrate chemiluminescence kit (Thermo Fisher Scientific).
Any kd mini protean gels
Manufactured by Bio-Rad
The Any-KD Mini-Protean gels are pre-cast polyacrylamide gels designed for protein electrophoresis. They feature a versatile polyacrylamide gradient that allows for the separation of a wide range of protein molecular weights in a single gel.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti il 1β antibody, by Novus Biologicals (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
2 protocols using any kd mini protean gels
1
Protein Expression Analysis by Western Blot
2
Comparative Photosynthetic Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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WT and mutants were grown in TAP to early log phase under very low light (0~2 μmol photons m-2 s-1). Total protein was solubilized as previously described [29 (link)] and extracted using the methanol/chloroform method [55 (link)]. Each sample was quantified for protein concentration using BCA protein assay (Pierce) to load 5 μg protein in each lane of pre-cast SDS-PAGE Any kD Mini-PROTEAN gels (Bio-Rad), except for the dilutions of the WT samples. Proteins were transferred from the gel to a polyvinylidene difluoride membrane (Immobilon-FL 0.45 μm, Millipore) via wet-transfer for immunodetection. The primary polyclonal antibodies raised against AtpB (AS16 3976), D1 DE-loop (AS10 704), CP43 (AS11 1787), PsaA (AS06 172), and PsaD (AS09 461) were obtained from Agrisera. Donkey anti-rabbit IgG antibody with horseradish peroxidase (1:10,000; GE Healthcare) was used as a secondary antibody and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) chemiluminescent system on a ChemiDoc MP imaging system (Bio-Rad).
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