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: 85 kDa
Observed band size: 85 kDa

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.

Immunofluorescence analysis of 4% paraformaldehyde fixed HeLa cells (Human epithelial cells from cervix adenocarcinoma) labeling Hsp90 beta with ab203085 at a 1/250 dilution showing cytoplasmic staining. Permeabilisation using 0.1% tritonX-100.

Secondary ab: Anti-Rabbit Alexa Fluor® 488 (ab150077) at 1/200 dilution. Counter stain is labeling tubulin (red) with ab7291 at 1/500 dilution with secondary antibody Anti-Mouse AlexaFluor® 594 (ab150120) at 1/400 dilution. DAPI stains the nucleus in blue. -ve control 1 is ab178854 at 1/250 dilution, Anti-Mouse AlexaFluor® 594 (ab150120) at 1/400 dilution. -ve control 2 is ab7291 at 1/500 dilution, Anti-Rabbit Alexa Fluor® 488 (ab150077) at 1/200 dilution.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Hsp90 beta with ab203085 at 1/500 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Counter stained with hematoxylin. Negative control also shown using PBS instead of primary antibody and secondary as above.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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: 85 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 [EPR16621] (ab203085) at 1/10000 dilution

Lane 1 : Hsp90 alpha recombinant protein fragment (GST-tag): aa533-732
Lane 2 : Hsp90 beta recombinant protein fragment (His-Tag®): aa525-724

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated)

Predicted band size: 85 kDa


Exposure time: 3 minutes

Blocking and Diluting buffer and concentration: 5%
NFDM/TBST.

ab203085 only recognizes Hsp90 beta based on recombinant protein WB result.

All lanes : Anti-Hsp90 beta antibody [EPR16621] (ab203085) at 1/20000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : 293T (Human epithelial cells from embryonic kidney) whole cell lysates
Lane 3 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates
Lane 4 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 85 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM /TBST

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

Lane 1 : Mouse brain
Lane 2 : Mouse heart
Lane 3 : Mouse kidney
Lane 4 : Mouse spleen
Lane 5 : Rat brain
Lane 6 : Rat heart
Lane 7 : Rat kidney
Lane 8 : Rat spleen
Lane 9 : C6 (Rat glial tumor cells) whole cell lysate
Lane 10 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 12 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 85 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM /TBST

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling Hsp90 beta with ab203085 at 1/500 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm and nucleus staining on neuron of cerebral cortex is observed. This protein is synthesized in cytoplasm, but it can shuttle between cytoplasm and nucleus depend on the cell status. Counter stained with hematoxylin. Negative control also shown using PBS instead of primary antibody and secondary as above.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse cerebral cortex tissue labeling Hsp90 beta with ab203085 at 1/500 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm and nucleus staining on neuron of cerebral cortex is observed. This protein is synthesized in cytoplasm, but it can shuttle between cytoplasm and nucleus depending on the cell status. Counter stained with hematoxylin. Negative control also shown using PBS instead of primary antibody and secondary as above.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex tissue labeling Hsp90 beta with ab203085 at 1/500 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm and nucleus staining on neuron of cerebral cortex is observed. This protein is synthesized in cytoplasm, but it can shuttle between cytoplasm and nucleus depending on the cell status. Counter stained with hematoxylin. Negative control also shown using PBS instead of primary antibody and secondary as above.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Hsp90 beta was immunoprecipitated from 1mg of Hela (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab203085 at 1/70 dilution. Western blot was performed of the immunoprecipitate using ab203085 at 1/5000 dilution. Goat Anti-Rabbit IgG, (H+L), peroxidase conjugated, was used as secondary antibody at 1:1000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM/TBST.

Flow cytometry analysis of 2% paraformaldehyde fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells using ab203085 at a 1/100 dilution (red) or a Rabbit monoclonal IgG (negative) (black). Goat anti rabbit IgG (FITC) secondary used at a 1/150 dilution.