Nzy supreme ecl hrp substrate

Proteins were harvested using a RIPA buffer (Merck, KGaA, Darmstadt, Germany). All the protein samples were diluted 1:1 in Laemmli buffer (Merck, KGaA, Darmstadt, Germany) and heated at 95 °C for 5 min before running the gel. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad Laboratories, Hercules, CA, USA). They were transferred to the nitrocellulose membrane through the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). Next, the membrane was processed and stained using the iBind Western system (Thermo Fisher Scientific, Waltham, MA, USA) and primary antibodies specific for CstF (1:2000 dilution of #SAB2700222, Sigma-Aldrich), ß-tubulin (1:4000 dilution of #ab6046, Abcam, Cambridge, UK), and horseradish peroxidase (HRP)-conjugated secondary antibody (1:4000 dilution of #1706515, Bio-Rad Laboratories, Hercules, CA, USA). The visualization of bands was performed through chemiluminescence using an NZY Supreme ECL HRP Substrate (NZYTech, Lisbon, Portugal) in a ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA). The band intensity was quantified using ImageLab software on USB drive #12012931 (Bio-Rad Laboratories, Hercules, CA, USA).

Total proteins were harvested using Laemmli buffer (Sigma-Aldrich) and heated at 95°C for 5 min. Samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 15% polyacrylamide gel and transferred to 0.2-μm pore nitrocellulose membrane (Amersham Protran, Cytiva). Membrane was blocked in 5% low-fat milk PBS with 0.1% Tween 20. Following blocking, the membrane was incubated in 1:2,000 dilution of primary antibodies specific for CstC (Cat # ABC20, Sigma-Aldrich) and β-tubulin (Cat # ab6046, Abcam) overnight. Membranes were washed in PBS-Tween and incubated with secondary horseradish peroxidase (HRP)-conjugated antibody (Cat # 1706515, Bio-Rad) for 1 h. Bands were visualized by chemiluminescence using NZY Supreme ECL HRP substrate (Cat # MB19301, NZYTech) in a ChemiDoc XRS (Bio-Rad). Quantification of band intensity was performed on ImageJ software.

Total proteins were harvested using a RIPA buffer (Merck, KGaA, Darmstadt, Germany). Prior to electrophoresis, the samples were diluted 1:1 in a Laemmli buffer (Merck, KGaA, Darmstadt, Germany) and heated at 95 °C for 5 min. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using TGX FastCast 10% acrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to the nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was processed and stained using the iBind Western system (Thermo Fisher Scientific, Waltham, MA, USA), and the antibodies specific for CstC (1:1000 dilution of #ab133495, Abcam, Cambridge, UK), β-tubulin (1:2000 dilution of #ab6046, Abcam, Cambridge, UK), and horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution of #1706515, Bio-Rad Laboratories, Hercules, CA, USA). The bands were visualized via chemiluminescence using an NZY Supreme ECL HRP substrate (NZYTech, Lisbon, Portugal) in an iBright CL1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). The quantification of band intensity was performed in Fiji software.