All lanes : Anti-Hsp90 antibody [H90-10] (ab58950) at 0.5 µg/ml

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : HSP90AB1 knockout HEK-293T cell lysate
Lane 3 : Saos-2 cell lysate
Lane 4 : HL-60 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 83 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab58950 observed at 85 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab58950 was shown to react with Hsp90 in wild-type HEK-293T cells in western blot with loss of signal observed in HSP90AB1 knockout cell line ab266117 (HSP90AB1 knockout cell lysate ab257190). Wild-type and HSP90AB1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab58950 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at 0.5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Hsp90 beta antibody [H90-10] (ab53497) at 1/5000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : HSP90AB1 knockout HEK-293T cell lysate
Lane 3 : Saos-2 cell lysate
Lane 4 : HL-60 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 83 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab53497 observed at 85 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab53497 was shown to react with Hsp90 beta in wild-type HEK-293T cells in western blot with loss of signal observed in HSP90AB1 knockout cell line ab266117 (HSP90AB1 knockout cell lysate ab257190). Wild-type and HSP90AB1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab53497 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Hsp90 beta antibody [E296] (ab32568) at 1/200000 dilution

Lane 2 : HSP90AB1 knockout HEK-293T cell lysate
Lane 3 : Saos-2 cell lysate
Lane 4 : HL-60 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 83 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab32568 observed at 85 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab32568 was shown to react with Hsp90 beta in wild-type HEK-293T cells in western blot with loss of signal observed in HSP90AB1 knockout cell line ab266117 (HSP90AB1 knockout cell lysate ab257190). Wild-type and HSP90AB1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab32568 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 200000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Hsp90 beta antibody [EPR16621] (ab203085) at 1/5000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : HSP90AB1 knockout HEK-293T cell lysate
Lane 3 : Saos-2 cell lysate
Lane 4 : HL-60 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 83 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab203085 observed at 85 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab203085 was shown to react with Hsp90 beta in wild-type HEK-293T cells in western blot with loss of signal observed in HSP90AB1 knockout cell line ab266117 (HSP90AB1 knockout cell lysate ab257190). Wild-type and HSP90AB1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab203085 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Hsp90 beta antibody [EPR16621] (ab203085) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : HSP90AB1 knockout HEK293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 83 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab203085 observed at 90 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab203085 Anti-Hsp90 beta antibody [EPR16621] was shown to specifically react with Hsp90 beta in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266117 (knockout cell lysate ab257190) was used. Wild-type and Hsp90 beta knockout samples were subjected to SDS-PAGE. ab203085 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 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 : Anti-Hsp90 beta antibody [E296] (ab32568) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : HSP90AB1 knockout HEK293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 83 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab32568 observed at 90 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab32568 Anti-Hsp90 beta antibody [E296] was shown to specifically react with Hsp90 beta in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266117 (knockout cell lysate ab257190) was used. Wild-type and Hsp90 beta knockout samples were subjected to SDS-PAGE. ab32568 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 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.

Homozygous: 5 bp deletion in exon3