Anti-HDAC6 antibody [EPR22951-29]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HDAC6 antibody [EPR22951-29] (AB239362)

Immunofluorescent analysis of 100% methanol fixed 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling HDAC6 with ab239362 at 1/200 (3 μg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody as secondary at 1/1000 (2 μg/ml) dilution. Confocal image showing cytoplasmic staining in Neuro-2a cell line (shown in green). The counterstain was ab7291 Anti-alpha Tubulin mouse monoclonal antibody used at 1/1000 (1μg/ml) followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) as secondary for the counterstain used at 1/1000 (2μg/ml) observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). Negative control 1 : ab239362 with ab150120 as secondary. Negative control 2 : ab7291 with ab150081 as secondary.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HDAC6 antibody [EPR22951-29] (AB239362)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Neuro-2a (Mouse neuroblastoma neuroblast) cells labeling HDAC6 with ab239362 at 1\600 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HDAC6 antibody [EPR22951-29] (AB239362)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labeling HDAC6 with ab239362 at 1\600 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

• IP

Unknown

Immunoprecipitation - Anti-HDAC6 antibody [EPR22951-29] (AB239362)

HDAC6 was immunoprecipitated from 0.35 mg NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10μg with ab239362 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab239362 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 whole cell lysate 10μg

Lane 2 : ab239362 IP in NIH/3T3 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab239362 in NIH/3T3 whole cell lysate

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-HDAC6 antibody [EPR22951-29] (ab239362)

Predicted band size: 131 kDa,47 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-HDAC6 antibody [EPR22951-29] (AB239362)

HDAC6 was immunoprecipitated from 0.35 mg mouse brain lysate 10μg with ab239362 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab239362 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse brain lysate 10μg

Lane 2 : ab239362 IP in mouse brain lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab239362 in mouse brain lysate

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

Exposure time : 3 mins.

All lanes:

Immunoprecipitation - Anti-HDAC6 antibody [EPR22951-29] (ab239362)

Predicted band size: 131 kDa

false

• WB

Lab

Western blot - Anti-HDAC6 antibody [EPR22951-29] (AB239362)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 18180281, 29201907, 28273454, 25108026).

Exposure time : Lane 1-2 : 32 seconds Lane 3 : 3 minutes.

All lanes:

Western blot - Anti-HDAC6 antibody [EPR22951-29] (ab239362) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate at 10 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 131 kDa

Observed band size: 130 kDa

false

• WB

Lab

Western blot - Anti-HDAC6 antibody [EPR22951-29] (AB239362)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-HDAC6 antibody [EPR22951-29] (ab239362) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

NIH/3T3 transfected with siRNA specifically targeting HDAC6, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 130 kDa,36 kDa

false

Exposure time: 4s

• WB

Lab

Western blot - Anti-HDAC6 antibody [EPR22951-29] (AB239362)

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

Kidney is a low-expression tissue.

A positive unidentified band between 15-20 kDa is detected in mouse kidney lysate.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 18180281, 29201907, 28273454, 25108026).

Exposure time : 48 seconds.

All lanes:

Western blot - Anti-HDAC6 antibody [EPR22951-29] (ab239362) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Rat brain tissue lysate at 20 µg

Lane 4:

Rat kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 131 kDa

Observed band size: 130 kDa

false