Samples were electrophoresed at 100 V for 1 h on a 16% (ubiquitin) or 10% (β-galactosidase) polyacrylamide gel in Tris-Glycine-SDS buffer using a Mini-PROTEAN 3 (Bio-Rad) apparatus. The gel was washed with Tris-Glycine buffer (Thermo Fisher) containing 20% methanol and transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) in Tris-Glycine buffer containing 20% methanol using a Mini-PROTEAN 3 (Bio-Rad) apparatus. The membrane was blocked for 2 h at room temperature with 1% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20 (BSA/TBS-T) and incubated overnight at 4 °C with the primary antibody, HRP-conjugated mouse monoclonal IgG1 [Ub Antibody (P4D1), sc-8017] diluted 1:500 with a BSA/ TBS-T mixture. The membrane was washed with TBS. Western blots were developed by incubating the membrane in Super-Signal West Pico Chemiluminescent Substrate (Thermo Scientific) for 5 min at room temperature, and the resulting chemiluminescence was imaged by using a Chem-iDoc XRS (Bio-Rad) instrument equipped with Quantity One software.
Tris glycine buffer
Manufactured by Thermo Fisher Scientific
Tris-Glycine buffer is a common buffer solution used in various biochemical and molecular biology applications. It is primarily used to maintain a stable pH environment during gel electrophoresis, Western blotting, and protein transfer processes. The buffer consists of a combination of Tris (tris(hydroxymethyl)aminomethane) and glycine, which work together to maintain a consistent pH range within the desired experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean 3, by Bio-Rad (1 mentions)
0.2 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Tris glycine buffer, by Bio-Rad (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Image studio lite v5, by LI COR (1 mentions)
2 protocols using tris glycine buffer
1
Western Blot Analysis of Ubiquitin and β-Galactosidase
2
Endocervical Tissue Explants HIV Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endocervical tissue explants were incubated with E2 (100 and 10,000 pg/ml) for 48 h. The explants were then either collected for WB analysis or challenged with 500 TCID50 HIV-1BaL. Explants challenged with HIV-1BaL were cultured for 7 days and collected for WB analysis.
The explants were washed with ice cold DPBS, snap frozen in liquid nitrogen, cut, and lysed in RIPA lysis and extraction buffer supplemented with protease inhibitor cocktail to prepare WCEs. Bradford assay was used to measure protein concentration. 60–120 µg of protein was mixed with NuPAGE LDS sample buffer and β-Mercaptoethanol and heated at 95 °C for 10–15 min. WCEs were resolved on NuPAGE 8–16% Tris Glycine gel in Tris Glycine buffer (Thermo Fisher scientific). The gel/blot transfer and Ab probing were done as described above.
Protein bands were quantified and normalized by β-actin using densitometry method (Image Studio Lite v5.2 software (LI-COR Biosciences)).
The explants were washed with ice cold DPBS, snap frozen in liquid nitrogen, cut, and lysed in RIPA lysis and extraction buffer supplemented with protease inhibitor cocktail to prepare WCEs. Bradford assay was used to measure protein concentration. 60–120 µg of protein was mixed with NuPAGE LDS sample buffer and β-Mercaptoethanol and heated at 95 °C for 10–15 min. WCEs were resolved on NuPAGE 8–16% Tris Glycine gel in Tris Glycine buffer (Thermo Fisher scientific). The gel/blot transfer and Ab probing were done as described above.
Protein bands were quantified and normalized by β-actin using densitometry method (Image Studio Lite v5.2 software (LI-COR Biosciences)).
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!