Anti-CDKL5 antibody [EPR26906-28]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CDKL5 antibody [EPR26906-28] (AB315220)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling CDKL5 with ab315220 at 1/50 (10.12 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic and nuclear staining in HepG2 cells. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CDKL5 antibody [EPR26906-28] (AB315220)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling CDKL5 with ab315220 at 1/500 dilution (0.1 ug, Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-CDKL5 antibody [EPR26906-28] (AB315220)

CDKL5 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab315220 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315220 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate

Lane 2 : ab315220 IP in HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab315220 in HepG2 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-CDKL5 antibody [EPR26906-28] (ab315220) at 1/30 dilution

All lanes:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 24s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CDKL5 antibody [EPR26906-28] (AB315220)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling CDKL5 with ab315220 at 1/50 (10.12 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic and nuclear staining in Neuro-2a cells. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• WB

Supplier Data

Western blot - Anti-CDKL5 antibody [EPR26906-28] (AB315220)

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

ab315220 detects both two isoforms of CDKL5, separately named as CDKL5a and CDKL5b, around 130 and 100 kDa in Western blot (PMID : 20861382).

All lanes:

Western blot - Anti-CDKL5 antibody [EPR26906-28] (ab315220) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 50 µg

Lane 2:

Mouse lung tissue lysate at 50 µg

Lane 3:

Mouse liver tissue lysate at 50 µg

Lane 4:

Rat brain tissue lysate at 50 µg

Lane 5:

Rat lung tissue lysate at 50 µg

Lane 6:

Rat lilver tissue lysate at 50 µg

Lane 7:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 50 µg

Lane 8:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 9:

C2C12 (mouse myoblast) whole cell lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 100 kDa,130 kDa,110 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-CDKL5 antibody [EPR26906-28] (AB315220)

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

ab315220 detects both two isoforms of CDKL5, separately named as CDKL5a and CDKL5b, around 130 and 100 kDa in Western blot (PMID : 20861382).

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

All lanes:

Western blot - Anti-CDKL5 antibody [EPR26906-28] (ab315220) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 50 µg

Lane 2:

HepG2 transfected with siRNA specifically targeting CDKL5 whole cell lysate at 50 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 4:

Human lung tissue lysate at 50 µg

Lane 5:

Human liver tissue lysate at 50 µg

Lane 6:

LNCaP (human prostate carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 7:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 100 kDa,130 kDa,110 kDa,36 kDa

false

Exposure time: 1s