After transfection to transiently express CFTR, transfection efficiency was assessed by preparing cell lysates for western blots. First, the media was aspirated and replaced with 300 μL (for a 6 well dish) of TNT Buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 1% Triton X-100) + 1 PIC. Plates were incubated on ice for 30 min with occasional agitation by hand before centrifugation at 13000 rpm (~16000×g) for 15 min at 4°C. The total protein concentration of the cleared lysates was determined as described above. Each cleared lysate was diluted into SDS Sample Buffer and volumes corresponding to 30 μg total protein were resolved by 5% polyacrylamide/SDS gels and SDS-PAGE. To assess CFTR expression, blots were treated with 1:2500 mouse anti-CFTR (antibody 217 from the Cystic Fibrosis Foundation Therapeutics antibody distribution program, University of North Carolina at Chapel Hill). All blots were washed with TBST as described above and then treated with 1:5000 horse anti-mouse-HRP secondary antibody in TBST (Cell Signaling Technology #7076) for 4 hr with ambient rocking. After secondary antibody treatment, the blots were again washed, developed with SuperSignal™ West Pico Chemiluminescent Substrate, and imaged using a Bio-Rad ChemiDoc™ XRS+Imager.
West pico chemiluminescent substrate
Manufactured by Bio-Rad
The West Pico Chemiluminescent Substrate is a reagent used for the detection of proteins in Western blot analysis. It produces a luminescent signal when it interacts with the HRP (Horseradish Peroxidase) enzyme, which is commonly used to label antibodies in Western blotting experiments.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using west pico chemiluminescent substrate
1
CFTR Transient Expression Analysis
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates were sonicated using a Heat Systems Ultrasonic Processor XL sonicator. Protein concentrations were determined using the DC Protein Assay kit and BioTek PowerWave XS2 plate reader. Lysates were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked with 5% BSA in 25 mM Tris, 150 mM sodium chloride, and 0.1% Tween-20 solution (TBS-T) rocking at room temperature for 1 h, then probed with the appropriate antibodies (anti-DRD2, DRD3, DRD4, PKC-θ, pWIP, WIP, pN-WASP, and N-WASP primary antibodies, 1:1000; anti-β-actin antibody (1:20,000); anti-α-tubulin antibody (1:10,000); and anti-GAPDH (1:2,500)) in 5% milk or BSA in TBS-T with 0.05% sodium azide with rocking at 4 °C overnight. Overnight incubation in primary antibody was followed by washing (3 × TBS-T) and incubation with the appropriate species-specific HRP antibody (1:10,000) in 5% milk in TBS-T with rocking at room temperature for 1 h. Western blots were developed using SuperSignal West Pico Chemiluminescent Substrate on a BioRad ChemiDoc MP. Densitometry was performed using FIJI and normalized to the housekeeping protein and control lysate proteins.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!