Infiltrated leaves expressing the protein of interest were powdered in a mortar under liquid N2. About 2 g of tissue was weighed, and to it, 3 volumes of lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM KCL, 1% Triton X100, Protease inhibitor 1 X [Roche], NEM 20uM) were added. The supernatant was collected after a spin at 16000g for 30 min and incubated with GFP-Trap (Chromtek) or MBP magnetic beads (NEB) for 3 h at 4 °C. Beads were magnetically separated from the lysate and washed 5 times in wash buffer (50 mM Tris-Cl, pH 7.4; 150 mM KCL, 1 mM PMSF) until the green colour completely disappeared. The final pull-down beads were transferred to a 1.5-ml tube and again washed twice with wash buffer. The 3X SDS sample dye was added to the beads, and the sample was heated at 70 °C for 10 min. The pull-down products were resolved in 4–20% Tris-Glycine SDS gradient gels (Bio-Rad). IP beads used are listed in Additional file 2 : Table S4.
Tris glycine sds gradient gels
Manufactured by Bio-Rad
Tris-Glycine SDS gradient gels are precast polyacrylamide gels used for the separation of proteins based on their molecular weight. They provide a gradient of polyacrylamide concentration, allowing for effective separation of a wide range of protein sizes. These gels are designed for use in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using tris glycine sds gradient gels
1
Immunoprecipitation of GFP- and MBP-tagged proteins
2
Immunoprecipitation of GFP-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Infiltrated leaves expressing the protein of interest were powdered in a mortar under liquid N 2 . About 2 g of tissue was weighed, and to it, 3 volumes of lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM KCL, 1 % Triton X100, Protease inhibitor 1 X [Roche], NEM 20uM) was added. The supernatant was collected after a spin at 16000 g for 30 min and incubated with GFP-Trap (Chromtek) or MBP magnetic beads (NEB) for 3 h at 4°C. Beads were magnetically separated from the lysate and washed 5 times in wash buffer (50 mM Tris-Cl, pH 7.4; 150 mM KCL, 1 mM PMSF) until the green colour completely disappeared. The final pull-down beads were transferred to a 1.5 ml tube and again twice with wash buffer. The 3X SDS sample dye was added to the beads and the sample was heated at 70°C for 10 min. The pull-down products were resolved in 4-20 % Tris-Glycine SDS gradient gels (Bio Rad). IP beads used are listed in Supplemental Table 2.
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!