To measure mitochondrial protein glutathionylation, mitochondrial lysates were separated by SDS-PAGE. Blots were probed for glutathionylation using a 1:1,000 dilution of the anti-GSH antibody (Virogen, Watertown, MA) followed by a 1:1,000 dilution of horseradish peroxidase-linked goat anti-mouse Ig secondary antibody (Cell Signaling). Blots were also probed with a 1:5,000 dilution of anti-Hsp60 (AbCam, Cambridge, MA) to control for equal loading. To visualize multiple bands on the same blot, blots were stripped with Restore Western Blot Stripping Buffer (Pierce) before being probed with a new antibody. Proteins were normalized to Hsp60 to correct for loading differences. To determine the overall level of protein glutathionylation, bands were quantified using a ScanJet 4C scanner (Hewlett Packard, Palo Alto, CA) and Image J analysis software (National Institutes of Health, Bethesda, MA). To measure Complex III protein glutathionylation, mitochondrial cell lysates were immunoprecipitated for Complex III using a Complex III Immunocapture kit (AbCam), separated by SDS PAGE and immunobloted with the anti-GSH antibody, as previously described [11 (link)].
Anti gsh antibody
Manufactured by Abcam
Sourced in United States
Anti-GSH antibody is a laboratory reagent used to detect and quantify the presence of glutathione (GSH) in biological samples. It functions by specifically binding to GSH, allowing for its identification and measurement through various analytical techniques.
Automatically generated - may contain errors
3 protocols using anti gsh antibody
1
Quantifying Mitochondrial Protein Glutathionylation
2
GSH Immunoprecipitation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2 mL H2O2 or PBS was added into the cell culture medium and incubated for 10 minutes. Cells were lysed, and anti‐GSH antibody (Abcam, USA) was then added to the cell lysate, which was incubated at 4°C for 12 hours. Next, agarose beads were added, and the reactions were incubated at 4°C for a further 2 hours. The supernatant was centrifuged, and the agarose beads were washed three times with PBS. Then, SDS loading buffer was added into a certain volume of PBS containing agarose beads. The mixture was denatured at 100°C for 10 minutes and then subjected to SDS‐PAGE as described below.
3
Probing Glutathionylation of NPM1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown at 107 were washed with PBS and scraped before being suspended in 1 ml PBS on ice. After centrifugation, the cell pellet was resuspended in 1 ml lysis buffer (50 mM Tris-HCl pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, plus cocktail inhibitor (Roche)) for 30 min gentle agitation at 4 °C, followed by sonication for two bursts of 10 s each at half power with a 2 mm stepped microtip (Sonics VCX-130, USA). After centrifugation, the supernatant was mixed with anti-NPM1 antibody (1: 200, PTG-lab), anti-GSH antibody (1: 100, Abcam) and anti-HDM2 antibody (1: 100, Calbiochem) overnight at 4 °C. Next, 30 μl of Protein A/G-Agarose (EMD Millipore) was added for a further 4 h agitation at 4 °C. Lastly, the resins were washed six times with 1 ml lysis buffer and then mixed with 2 × SDS sample buffer, boiled 10 min and analysed by western blotting. Specifically for glutathionylated NPM1 samples, β-mercaptoethanol was not included in the 2 × SDS sample buffer.
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!