Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] - BSA and Azide free (AB228457)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Smad1 (phospho S463 + S465) with ab226821 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Nuclear staining in hBMP2-treated NIH/3T3 cells. Cells were FBS-deprived overnight before treatment with 50 ng/ml hBMP2 for 30 minutes.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab226821).

• IP

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Immunoprecipitation - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] - BSA and Azide free (AB228457)

Smad 1 (phospho S463 + S465) was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryo fibroblast cell line) grown in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate with ab226821 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab226821 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : NIH/3T3 grown in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate 10 μg (Input).

Lane 2 : ab226821 IP in NIH/3T3 grown in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab226821 in NIH/3T3 grown in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate.

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

Exposure time : 10 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab226821).

All lanes:

Immunoprecipitation - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] (<a href='/en-us/products/primary-antibodies/smad1-phospho-s463-s465-antibody-epr20662-20-ab226821'>ab226821</a>)

Predicted band size: 52 kDa

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• Dot

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Dot Blot - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] - BSA and Azide free (AB228457)

Dot blot analysis of Smad1 (phospho S463 + S465) labeled with ab226821 at 1/1000 dilution.

Lane 1 : Smad1 (phospho S463/S465) peptide;

Lane 2 : Smad1 (phospho S463) peptide;

Lane 3 : Smad1 (phospho S465) peptide;

Lane 4 : Smad1 peptide (not phosphorylated);

Lane 5 : Smad5 (phospho S463/S465) peptide;

Lane 6 : Smad5 (phospho S463) peptide;

Lane 7 : Smad5 (phospho S465) peptide;

Lane 9 : Smad5 peptide (not phosphorylated).

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

Based on sequence homology, this antibody cross-reacts with Smad5 (phospho S463/S465) and Smad9 (phospho S465/S467).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab226821).