Anti-Hsp90 alpha 抗体 [68/Hsp90] (ab282108)

All lanes : Anti-Hsp90 alpha antibody [68/Hsp90] (ab282108) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : HSP90AA1 KO HEK293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (IRDye® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (ab216777) at 1/10000 dilution

Observed band size: 83 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lanes 1-3: Merged signal (red and green). Green - ab282108 observed at 83KDa. Red - loading control ab181602 (Rabbit monoclonal [EPR16891] to GAPDH) observed at 36 kDa.

Lanes 1-2: ab282108 Anti-Hsp90 alpha antibody was shown to react with Hsp90 alpha in HEK293T cells in Western blot. Loss of signal was observed when HSP90AA1 knockout sample was used. Wild-type and
HSP90AA1 knockout samples were subjected to SDS-PAGE.

ab282108 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated at 4? overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680CW) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800RD) preadsorbed (ab216772) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling Hsp90 alpha with ab282108 at 1/50 (19.98 ug/ml) dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in 293T cells ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

All lanes : Anti-Hsp90 alpha antibody [68/Hsp90] (ab282108) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate
Lane 2 : K-562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Observed band size: 83 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 7 seconds

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling Hsp90 alpha with ab282108 at 1/50 (19.98 ug/ml) dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in HeLa cells ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.