Lens cell lysate samples were mixed with Laemmli buffer (Sigma-Aldrich) and were heated to 95° C for 10 minutes. After resolution with Mini Protean TGX Precast gel 4–15%, proteins from gels were transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The following primary antibodies were used for immunodetection: mouse anti-human AR (1:1000; Santa Cruz, TX, USA), mouse anti-actin (1:1000; Sigma-Aldrich). For detection of extracellular signal-related kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases (JNK), we used rabbit anti-phospho ERK1/2 (1:1000; Cell Signaling, MA, USA), rabbit anti-ERK1/2 (1:1000; Cell Signaling), rabbit anti-phospho JNK (1:1000; Cell Signaling), and rabbit anti-JNK (1:1000; Cell Signaling), respectively. Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase (1:2000; Millipore, Bedford, MA, USA), as well as the Immun-Star WesternC Kit (Bio-Rad, CA, USA) were used to detect chemilluminescence using a BioRad ChemiDoc XRS+ imaging system.
Mini protean tgx precast gel
Manufactured by Cytiva
Sourced in United States
The Mini Protean TGX Precast gel is a laboratory equipment designed for protein electrophoresis. It is a pre-cast polyacrylamide gel that is ready-to-use, providing a convenient solution for SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) analysis of protein samples.
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Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Rabbit anti erk1 2, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Laemmli buffer, by Merck Group (1 mentions)
Immun star westernc kit, by Bio-Rad (1 mentions)
Rabbit anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Rabbit anti jnk, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho jnk, by Cell Signaling Technology (1 mentions)
Western breeze chemiluminescent western blot immunodetection kit, by Thermo Fisher Scientific (1 mentions)
Protran, by Cytiva (1 mentions)
2 protocols using mini protean tgx precast gel
1
Western Blot for Protein Analysis
2
Western Blot Analysis of GFP Proteins
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Proteins were extracted from 2-week-old seedlings cultured on 0.5× MS medium using the nuclei lysis buffer as mentioned above. Protein samples were separated on Bio-Rad Mini-PROTEAN® TGX™ Precast Gel and transferred onto Amersham™ Protran® western blotting membrane (nitrocellulose, pore size: 0.2 μm). Subsequent steps were performed following instructions for ‘WesternBreeze® Chemiluminescent Western Blot Immunodetection’ Kit (Invitrogen). The GFP antibody used is a mixture of two monoclonal antibodies from mouse IgG1κ (clone 7.1 and 13.1, Sigma-Aldrich) at a 1:1000 dilution. An anti-actin antibody, a rabbit polyclonal antibody targeting a set of actins in Arabidopsis (Agrisera) was used as a control (at 1:2500 dilution). The protein amounts were quantified using Quantity One software (v4.6.2.70). Uncropped scans for western blot membranes were included in the Source Data file or Supplementary Information file.
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