Anti-SIK3 antibody [EPR23554-71]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIK3 antibody [EPR23554-71] (AB255701)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labelling SIK3 with ab255701 at 1/100 (5.85 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in human ovarian cancer (PMID : 21399663). The section was incubated with ab255701 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIK3 antibody [EPR23554-71] (AB255701)

Immunohistochemical analysis of paraffin-embedded Human bladder cancer tissue labelling SIK3 with ab255701 at 1/100 (5.85 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in human bladder cancer. The section was incubated with ab255701 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SIK3 antibody [EPR23554-71] (AB255701)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Neuro-2a (Mouse neuroblastoma neuroblast) cells labelling SIK3 with ab255701 at 1/50 dilution (1ug) (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). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SIK3 antibody [EPR23554-71] (AB255701)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells lebelling SIK3 with ab255701 at 1/50 (11.7 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2ug/mL) dilution (Green). Confocal image showing cytoplasmic staining in Neuro-2a cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5ug/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 (2ug/mL) dilution.

• IP

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Immunoprecipitation - Anti-SIK3 antibody [EPR23554-71] (AB255701)

SIK3 was immunoprecipitated from 0.35 mg Mouse brain tissue lysate 10 ug with ab255701 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab255701 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse brain tissue lysate 10 ug

Lane 2 : ab255701 IP in Mouse brain tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab255701 in Mouse brain tissue lysate

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

Exposure time : 41 seconds

All lanes:

Immunoprecipitation - Anti-SIK3 antibody [EPR23554-71] (ab255701)

Predicted band size: 140 kDa

false

• WB

Lab

Western blot - Anti-SIK3 antibody [EPR23554-71] (AB255701)

Blocking buffer and concentration : 5% NFDM/TBST

Exposure time :

Lane 1 : 3 minutes
Lane 2 & 3 : 26 seconds

The expression profile/molecular weight observed is consistent with that described in the literature (PMID : 32542037).

All lanes:

Western blot - Anti-SIK3 antibody [EPR23554-71] (ab255701) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 60 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 140 kDa

Observed band size: 69 kDa,73 kDa

false

• WB

Lab

Western blot - Anti-SIK3 antibody [EPR23554-71] (AB255701)

Blocking / Dilution buffer and concentration : 5% NFDM/TBST

The expression profile/molecular weight observed is consistent with that described in the literature (PMID : 32542037).
Low expression : NIH : OVCAR-3 (PMID : PMID : 31762812)

All lanes:

Western blot - Anti-SIK3 antibody [EPR23554-71] (ab255701) at 1/1000 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

NIH:OVCAR-3 (human ovary adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate at 20 µg

Lane 4:

C6 (rat glial tumor glial cell), whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 140 kDa

Observed band size: 69 kDa,73 kDa

false

Exposure time: 3min