Antibodies, Proteins, Kits and Reagents for Life Science

311 587 ₸ Product size

100 µl 311 587 ₸

Order now and get it on Tuesday March 02, 2021

Anti-Hsp90 beta antibody [E296] (ab32568)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [E296] to Hsp90 beta
Suitable for: WB, IHC-P, ICC/IF
Knockout validated
Reacts with: Mouse, Rat, Human

Product name

Anti-Hsp90 beta antibody [E296]
See all Hsp90 beta primary antibodies
Description

Rabbit monoclonal [E296] to Hsp90 beta
Host species

Rabbit

Tested Applications & Species

Application	Species
ICC/IF	Human
IHC-P	Human
WB	Mouse Human

See all applications and species data

Immunogen

Synthetic peptide within Human Hsp90 beta aa 1-100 (N terminal). The exact sequence is proprietary.
Positive control
- WB: Saos-2, HL-60, HEK293T, Jurkat, SH-SY5Y, Raji, A431 and HeLa whole cell lysate (ab150035); Mouse brain and heart tissue lysates; Rat brain and heart tissue lysates. IHC-P: Stomach and urinary bladder carcinoma tissues. ICC/IF: HepG2 cells.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Form

Liquid
Storage instructions

Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
Storage buffer

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

E296
Isotype

IgG
Research areas

Western blot - Anti-Hsp90 beta antibody [E296] (ab32568)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp90 beta antibody [E296] (ab32568)

Immunohistochemical staining of paraffin embedded human stomach with purified ab32568 at a working dilution of 1 in 150. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
Immunocytochemistry/ Immunofluorescence - Anti-Hsp90 beta antibody [E296] (ab32568)

Immunofluorescence staining of HepG2 cells with purified ab32568 at a working dilution of 1 in 100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 555 goat anti rabbit (ab150082), used at a dilution of 1 in 400. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab32568 was used at a dilution of 1/200 followed by an Alexa Fluor^® 488 goat anti-mouse antibody at a dilution of 1/500.
Western blot - Anti-Hsp90 beta antibody [E296] (ab32568)

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

All lanes : Anti-Hsp90 beta antibody [E296] (ab32568) at 1/100000 dilution (purified)

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart tissue lysate
Lane 3 : Rat brain tissue lysate
Lane 4 : Rat heart tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
Western blot - Anti-Hsp90 beta antibody [E296] (ab32568)

Anti-Hsp90 beta antibody [E296] (ab32568) at 1/100000 dilution (purified) + SH-SH5Y cell lysate at 20 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 83 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?
Western blot - Anti-Hsp90 beta antibody [E296] (ab32568)

All lanes : Anti-Hsp90 beta antibody [E296] (ab32568) at 1/100000 dilution (purified)

Lane 1 : HeLa cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Raji cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
Western blot - Anti-Hsp90 beta antibody [E296] (ab32568)

Anti-Hsp90 beta antibody [E296] (ab32568) at 1/500 dilution (unpurified) + Hela cell lysate

Predicted band size: 83 kDa
Observed band size: 92 kDa why is the actual band size different from the predicted?
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp90 beta antibody [E296] (ab32568)

Immunohistochemical analysis of paraffin-embedded human urinary bladder carcinoma using unpurified ab32568 at 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Anti-Hsp90 beta antibody [E296] (ab32568)

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