Anti-Paxillin 抗体 [Y113]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin antibody [Y113] (AB32084)

Immunohistochemistry analysis of paraffin-embedded human breast carcinoma tissue sections labelling Paxillin with ab32084 at 1/1200 dilution. The section was incubated with ab32084 for 30 mins at room temperature. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.
Positive staining on human breast carcinoma tissue. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin antibody [Y113] (AB32084)

Immunohistochemistry analysis of paraffin-embedded human cerebrum tissue sections labelling Paxillin with ab32084 at 1/1200 dilution. The section was incubated with ab32084 for 30 mins at room temperature. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.
Positive staining on human cerebrum tissue. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [Y113] (AB32084)

Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Paxillin with purified ab32084 at 1/50 dilution (2.88 μg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Paxillin antibody [Y113] (AB32084)

Intracellular Flow Cytometry analysis ofHeLa (human cervix adenocarcinoma) cells labeling with purified ab32084 at 1/100 dilution (10μg/ml) (Red). Cells were fixed with4% paraformaldehydeand permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody.Rabbit monoclonal IgG (Black) (ab172730) was used as a isotype control.Cell without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

• IP

Lab

Immunoprecipitation - Anti-Paxillin antibody [Y113] (AB32084)

Purified ab32084 at 1/20 dilution (0.7μg) immunoprecipitating Paxillin in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg.
Lane 2 (+) : ab32084 + HeLa whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32084 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 48 kDa

All lanes:

Immunoprecipitation - Anti-Paxillin antibody [Y113] (ab32084)

Predicted band size: 65 kDa

Observed band size: 68 kDa

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• WB

Lab

Western blot - Anti-Paxillin antibody [Y113] (AB32084)

Anti-PXN antibody [Y113] (ab32084) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32084 was shown to bind specifically to PXN. A band was observed at 70 kDa in wild-type A431 cell lysates with no signal observed at this size in PXN knockout cell line ab261892 (knockout cell lysate ab261701). To generate this image, wild-type and PXN knockout A431 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween$®$ 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

All lanes:

Western blot - Anti-Paxillin antibody [Y113] (ab32084) at 1/1000 dilution

Lane 2:

Western blot - Human PXN (Paxillin) knockout A-431 cell lysate (<a href='/products/cell-lysates/human-pxn-paxillin-knockout-a-431-cell-lysate-ab261701'>ab261701</a>)

Lane 2:

Western blot - Human PXN (Paxillin) knockout A-431 cell line (<a href='/products/cell-lines/human-pxn-paxillin-knockout-a-431-cell-line-ab261892'>ab261892</a>)

Predicted band size: 65 kDa

Observed band size: 70 kDa

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• WB

Unknown

Western blot - Anti-Paxillin antibody [Y113] (AB32084)

All lanes:

Western blot - Anti-Paxillin antibody [Y113] (ab32084) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

Lane 2:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Lane 3:

Mouse heart lysate

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 65 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Paxillin antibody [Y113] (AB32084)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• WB

CiteAb

Western blot - Anti-Paxillin antibody [Y113] (AB32084)

Western Blotting using Anti-Paxillin antibody [Y113], ab32084. Publication image from Yang, J. et al., 2022, Nat Commun, 35488005. Legend direct from paper.

Paxillin binds to full-length active talin through multiple sites.a Paxillin prefers binding to activated talin as shown by GST pulldown. GST-tagged full-length paxillin was immobilized to GST beads and incubated with the same amount of full-length wild-type talin (talinFL WT) and full- length activated talin (talinFL TM). Pull-down fraction of talin was detected by talin antibody on western blot. Immobilized GST proteins were detected by GST antibody on western blot after 10x dilution. Four independent experiments were performed. b N-terminal part of paxillin (paxi1–160) is mainly responsible for binding to full- length active talin, while paxillin C-terminal (paxi161–605) shows no binding to talinFL TM. Pull-down fraction of talin was detected by Anti-6xHis antibody on western blot. Immobilized GST protein was resolved by SDS-PAGE and shown by Coomassie blue staining. As can be recognized from Coomassie staining, GST-paxillin 161–605 became unstable upon the 1–160 deletion, which degraded further after gel filtration. To alleviate this problem, we loaded much more paxillin 161–605 to have the major band (see arrow) to match other paxillin inputs, yet there was still no talin pulled down, indicating that paxillin 161–605 does not contribute to talin binding in contrast to paxillin 1–160. Four independent experiments were performed. c The HSQC spectra of 50 µM 15N-labeled talin-R2 in the absence (black) and presence of 100 µM unlabeled paxillin-γ 1–160 (red) showing direct interaction between paxillin and talin R2. d Cartoon representation of the best model of Paxillin-LD2 (in cyan) and talin-R2 (in green) complex through Haddock calculation. Side chains of critical residues involved in the binding are displayed and labeled. Red-dashed lines indicate potential H-bonds. e Mutations in talin-R2 (A680E/K687E, AKEE) abolished talin-R2 binding to paxillin 1–160 at the same experimental condition as (c). f Summary of mutation testing results from HSQC. Uncropped images are provided in Source Data file.

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• WB

CiteAb

Western blot - Anti-Paxillin antibody [Y113] (AB32084)

Western Blotting using Anti-Paxillin antibody [Y113], ab32084. Publication image from Yang, J. et al., 2022, Nat Commun, 35488005. Legend direct from paper.

Paxillin binds to full-length active talin through multiple sites.a Paxillin prefers binding to activated talin as shown by GST pulldown. GST-tagged full-length paxillin was immobilized to GST beads and incubated with the same amount of full-length wild-type talin (talinFL WT) and full- length activated talin (talinFL TM). Pull-down fraction of talin was detected by talin antibody on western blot. Immobilized GST proteins were detected by GST antibody on western blot after 10x dilution. Four independent experiments were performed. b N-terminal part of paxillin (paxi1–160) is mainly responsible for binding to full- length active talin, while paxillin C-terminal (paxi161–605) shows no binding to talinFL TM. Pull-down fraction of talin was detected by Anti-6xHis antibody on western blot. Immobilized GST protein was resolved by SDS-PAGE and shown by Coomassie blue staining. As can be recognized from Coomassie staining, GST-paxillin 161–605 became unstable upon the 1–160 deletion, which degraded further after gel filtration. To alleviate this problem, we loaded much more paxillin 161–605 to have the major band (see arrow) to match other paxillin inputs, yet there was still no talin pulled down, indicating that paxillin 161–605 does not contribute to talin binding in contrast to paxillin 1–160. Four independent experiments were performed. c The HSQC spectra of 50 µM 15N-labeled talin-R2 in the absence (black) and presence of 100 µM unlabeled paxillin-γ 1–160 (red) showing direct interaction between paxillin and talin R2. d Cartoon representation of the best model of Paxillin-LD2 (in cyan) and talin-R2 (in green) complex through Haddock calculation. Side chains of critical residues involved in the binding are displayed and labeled. Red-dashed lines indicate potential H-bonds. e Mutations in talin-R2 (A680E/K687E, AKEE) abolished talin-R2 binding to paxillin 1–160 at the same experimental condition as (c). f Summary of mutation testing results from HSQC. Uncropped images are provided in Source Data file.

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