Anti-Glutathione Synthetase antibody [EPR6563]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Glutathione Synthetase antibody [EPR6563] (AB133592)

Overlay histogram showing Jurkat cells stained with ab133592 (red line). The cells were fixed with 80% methanol (5 min)/ and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab133592, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Synthetase antibody [EPR6563] (AB133592)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling Glutathione Synthetase with ab133592 at 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-Glutathione Synthetase antibody [EPR6563] (AB133592)

Lanes 1- 4 : Merged signal (red and green). Green - ab133592 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab133592 was shown to react with GSS in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266342 (knockout cell lysate ab257460) was used. Wild-type HEK-293T and GSS knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab133592 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Glutathione Synthetase antibody [EPR6563] (ab133592) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

GSS knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human GSS (Glutathione Synthetase) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-gss-glutathione-synthetase-knockout-hek-293t-cell-line-ab266342'>ab266342</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Predicted band size: 52 kDa

Observed band size: 50 kDa

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• WB

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Western blot - Anti-Glutathione Synthetase antibody [EPR6563] (AB133592)

All lanes:

Western blot - Anti-Glutathione Synthetase antibody [EPR6563] (ab133592) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

HT-1080 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

Predicted band size: 52 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-Glutathione Synthetase antibody [EPR6563] (AB133592)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.