Anti-Paxillin (phospho S126) antibody

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin (phospho S126) antibody (AB24402)

Immunohistochemical analysis of human breast carcinoma labeling Paxillin (phospho S126) with ab24402 at 1/20 dilution (right) compared to a negative control without primary antibody (left).

To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab24402 diluted in 3% BSA-PBS at a dilution of 1/20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Paxillin (phospho S126) antibody (AB24402)

Immunofluorescence analysis of 70% confluent log phase NIH/3T3 cells treated with 0.5 ug of PDGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. Labeling Paxillin (phospho S126) with ab24402 at 1/250 dilution in 0.1% BSA and incubated for 3 hours at room temperature. Followed by Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1/2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Rhodamine Phalloidin at 1/300 dilution. Panel d is a merged image showing punctuated nuclear localization. Panel e is untreated cell with no signal. Panel f is a no primary antibody control. The images were captured at 60X magnification.

• WB

Supplier Data

Western blot - Anti-Paxillin (phospho S126) antibody (AB24402)

The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate

All lanes:

Western blot - Anti-Paxillin (phospho S126) antibody (ab24402) at 1/1000 dilution

Lane 1:

A549 (Human lung carcinoma cell line) whole cell lysate at 30 µg

Lane 2:

A549 whole cell lysate treated with 0.1 ug/mL of HGF treatment for 10 minutes at 30 µg

Lane 3:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 30 µg

Lane 4:

HepG2 whole cell lysate with 0.1 ug/mL of HGF treatment for 10 minutes at 30 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate at 1/1000 dilution

Predicted band size: 65 kDa

Observed band size: 68 kDa

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• WB

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Western blot - Anti-Paxillin (phospho S126) antibody (AB24402)

Lysates were resolved on a 10% polyacrylamide gel and transferred to PVDF. The membrane was blocked with a 5% milk-TBST buffer for one hour at room temperature, and then incubated with the Paxillin [pS¹²⁶] antibody for two hours at room temperature in a 1% milk-TBST buffer, following its prior incubation with or without peptides. Following incubation with goat F(ab’)₂ anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that only the phosphopeptide corresponding to Paxillin [pS 126 ] blocks the antibody signal, thereby demonstrating the specificity of the antibody. No competition was seen following incubation with paxillin phosphopeptides to S¹³⁰, S¹⁷⁸, S³⁸⁰, S⁴⁵⁵, or S⁴⁷⁹ (not shown). The antibody was also shown to be specific using 293 cells transfected with wild-type EGFP-tagged human paxillin treated with EGF (not shown).

All lanes:

Western blot - Anti-Paxillin (phospho S126) antibody (ab24402) at 1/1000 dilution

Lane 1:

Lysate from untreated RAW 264.7 cells

Lane 2:

Lysate from RAW 264.7 cells treated with 1 µg/mL LPS for 60 minutes

Lane 3:

Lysate from RAW 264.7 cells treated with 1 µg/mL LPS for 60 minutes with non-phosphorylated peptide corresponding to the immunogen

Lane 4:

Lysate from RAW 264.7 cells treated with 1 µg/mL LPS for 60 minutes with generic phosphoserine-containing peptide

Lane 5:

Lysate from RAW 264.7 cells treated with 1 µg/mL LPS for 60 minutes with phosphopeptide corresponding to Paxillin [pS⁹¹ ]

Lane 6:

Lysate from RAW 264.7 cells treated with 1 µg/mL LPS for 60 minutes with phosphopeptide immunogen

Secondary

All lanes:

Goat F(ab)₂ anti-rabbit IgG HRP conjugate.

Predicted band size: 65 kDa

Observed band size: 68 kDa

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