Anti-SIK3 (phospho S551) antibody [EPR20878-12] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SIK3 (phospho S551) antibody [EPR20878-12] - BSA and Azide free (AB283510)

This data was developed using ab225634, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (Human embryonic kidney epithelial cell) cells transfected with flag-tagged SIK3 WT expression vector cells labelling SIK3 (phospho S551) with ab225634 at 1/500 dilution (0.1 μg) (Right) compared with a Rabbit monoclonal IgG isotype control (ab172730) (Left). A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SIK3 (phospho S551) antibody [EPR20878-12] - BSA and Azide free (AB283510)

This data was developed using ab225634, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T cells labelling SIK3 (phospho S551) with ab225634 at 1/100 (4.61 μg/mL) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody (ab150077) at 1/1000 (2 μg/mL) dilution (Green). Confocal image showing cytoplasmic staining in HEK-293T cells transfected with flag-tagged SIK3 WT expression vector, and no staining in HEK-293T cells transfected with flag-tagged SIK3 T551A expression vector. Anti-FLAG mouse monoclonal antibody was used to counterstain tubulin at 1/500 (2 μg/mL) dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 (2 μg/mL) dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIK3 (phospho S551) antibody [EPR20878-12] - BSA and Azide free (AB283510)

This data was developed using ab225634, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labelling SIK3 (phospho S551) with ab225634 at 1/1000 (0.461 μg/mL) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on mouse cerebral cortex without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab225634 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-Fr

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Immunohistochemistry (Frozen sections) - Anti-SIK3 (phospho S551) antibody [EPR20878-12] - BSA and Azide free (AB283510)

This data was developed using ab225634, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum (fresh) tissue labeling SIK3 (phospho S551) with ab225634 at 1/100 (4.61 μg/mL) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution (Green). Positive staining on mouse cerebrum, and no staining has been shown after phosphatase treatment at 37°C for 2 hours. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• WB

Collaborator

Western blot - Anti-SIK3 (phospho S551) antibody [EPR20878-12] - BSA and Azide free (AB283510)

This data was developed using ab225634, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 3% BSA/TBST.

Data was kindly provided by Dr. Qinghua Liu, NIBS.

The 1st Ab working concentration was 5 μg/ml.

All lanes:

Western blot - Anti-SIK3 (phospho S551) antibody [EPR20878-12] (<a href='/en-us/products/primary-antibodies/sik3-phospho-s551-antibody-epr20878-12-ab225634'>ab225634</a>) at 0.461 µg/mL

All lanes:

Mouse cortex tissue lysate at 4 µg

Predicted band size: 140 kDa,70 kDa

Observed band size: 150 kDa,70 kDa

false

Data was kindly provided by Dr. Qinghua Liu, NIBS.

• WB

Lab

Western blot - Anti-SIK3 (phospho S551) antibody [EPR20878-12] - BSA and Azide free (AB283510)

This data was developed using ab225634, the same antibody clone in a different buffer formulation.

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

This blot was developed using a higher sensitivity ECL substrate.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-SIK3 (phospho S551) antibody [EPR20878-12] (<a href='/en-us/products/primary-antibodies/sik3-phospho-s551-antibody-epr20878-12-ab225634'>ab225634</a>) at 1/1000 dilution

Lane 1:

Mouse hippocampus tissue lysate (Untreated membrane) at 10 µg

Lane 2:

Mouse hippocampus tissue lysate (Phosphatase treated membrane) at 10 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 1/20000 dilution

Predicted band size: 140 kDa

Observed band size: 70 kDa

false

• Dot

Supplier Data

Dot Blot - Anti-SIK3 (phospho S551) antibody [EPR20878-12] - BSA and Azide free (AB283510)

This data was developed using ab225634, the same antibody clone in a different buffer formulation.

Dot blot analysis of SIK3 (phospho S551) using ab225634 at 1/1000 (0.461 μg/mL) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

Lane 1 : SIK3 (phospho S551) peptide 1

Lane 2 : SIK3 (phospho S551) peptide 2

Lane 3 : SIK3 non-phospho peptide

Exposure time : 3 minutes.

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