Aliquots (40 µL) of each soluble mating secretome were prepared for SDS-PAGE by mixing with 4× Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and denaturation at 55 °C for 5 min. Each sample was then fractionated on a Criterion TGX 4–15% gradient gel (Bio-Rad, Hercules, CA, USA). Electrophoresis of the first set of samples was stopped when the dye band had traveled 3 cm, and the gel was stained with QC Colloidal Coomassie (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s protocol. Based on molecular weight standards analyzed at the same time, each lane was cut into 10 slices that covered material migrating from the dye band to the top of the gel. Electrophoresis of the second set of samples was stopped when the dye band had traveled 2.5 cm; after staining with QC Colloidal Coomassie, each lane was cut into 4 slices covering the entire molecular weight range. Gel slices were stored frozen in microfuge tubes before preparation for LC-MS/MS analysis after in-gel digestion with trypsin.
Criterion tgx 4 15 gradient gel
Manufactured by Bio-Rad
Sourced in United States
The Criterion TGX 4–15% gradient gel is a pre-cast polyacrylamide gel used for protein separation and analysis. It features a 4–15% polyacrylamide gradient, which allows for the separation of a wide range of protein molecular weights. The gel is designed for use in Criterion electrophoresis systems.
Automatically generated - may contain errors
2 protocols using criterion tgx 4 15 gradient gel
1
Proteomic Analysis of Mating Secretome
2
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ten micrograms of protein from cell lysates or 0.1 μg of recombinant protein was suspended in Laemmli buffer and heated to 95°C for 10 min for SDS-PAGE, or suspended in SDS-free sample buffer for non-denaturing PAGE. Proteins were separated on 10% SDS-polyacrylamide or non-denaturing polyacrylamide gels, except in Fig. 3 A where proteins were separated on a pre-cast Bio-Rad Criterion™ TGX™ 4–15% gradient gel. After transfer of proteins to polyvinylidene difluoride (PVDF) membranes (Millipore) and blocking in 3% (w/v) powdered skimmed milk in Tris-buffered saline/0.1% Tween 20 (TBS-T), membranes were incubated overnight at 4°C in primary antibody [mouse anti-alpha synuclein 211 (Abcam) or mouse anti-amyloid β 4G8 (Covance)] diluted in blocking solution. After washing three times in TBS-T, the membrane was incubated with secondary antibody for 1 h at room temperature. The membrane was washed again in TBS-T and the signal visualized with ECL reagent (Millipore) and exposure in the Bio-Rad Gel Doc™.
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!