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Purified Mouse Anti-Paxillin
Purified Mouse Anti-Paxillin

Western blot analysis of Paxillin on a human endothelial lysate. Lane 1: 1:1000, lane 2: 1:2000, lane 3: 1:4000 dilution of the anti-Paxillin antibody.

Purified Mouse Anti-Paxillin

Immunofluorescent staining of HeLa (ATCC CCL-2) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~10 000 cells per well.  After overnight incubation, cells were stained using the alcohol perm protocol and the anti-Paxillin antibody. The second step reagent was Alexa Fluor® 555 conjugated anti-mouse Ig (Invitrogen). The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective. This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Western blot analysis of Paxillin on a human endothelial lysate. Lane 1: 1:1000, lane 2: 1:2000, lane 3: 1:4000 dilution of the anti-Paxillin antibody.

Immunofluorescent staining of HeLa (ATCC CCL-2) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~10 000 cells per well.  After overnight incubation, cells were stained using the alcohol perm protocol and the anti-Paxillin antibody. The second step reagent was Alexa Fluor® 555 conjugated anti-mouse Ig (Invitrogen). The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective. This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing), Mouse, Rat, Dog, Chicken, Cow (Tested in Development)
Mouse IgG1
Chicken Paxillin aa. 1-557
Western blot (Routinely Tested), Bioimaging (Tested During Development), Immunohistochemistry, Immunoprecipitation (Not Recommended)
68 kDa
250 µg/ml
AB_397918
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

Bioimaging

1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.

2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well.  Incubate for 10 minutes at room temperature (RT).

3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:

a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.

     OR

b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.

5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.

6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.

7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.

8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.

9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.

10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

11. View and analyze the cells on an appropriate imaging instrument.

Bioimaging:  For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp

Western blot:  For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Triton is a trademark of the Dow Chemical Company.
610569 Rev. 2
Antibody Details
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177/Paxillin

Paxillin, a focal adhesion protein, is a substrate for several tyrosine kinases such as src, FAK, and p120BRC/ABL. The tyrosine phosphorylation of paxillin is affected by conditions that change cell-cell adhesion. This is consisent with the possibility that paxillin is involved in the regulation of cell morphology. Additionally, because of its SH3-binding domain, paxillin associates tightly with FAK and Crk in an extracellular matrix-independent manner. Although paxillin was initially detected in fibroblasts, its phosphorylation may be important during neurite extension during differentiation.

610569 Rev. 2
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
610569 Rev.2
Citations & References
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Development References (5)

  1. Ku H, Meier KE. Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells. J Biol Chem. 2000; 275(15):11333-11340. (Biology: Immunoprecipitation, Western blot). View Reference
  2. Leventhal PS, Feldman EL. Tyrosine phosphorylation and enhanced expression of paxillin during neuronal differentiation in vitro. J Biol Chem. 1996; 271(11):5957-5960. (Biology). View Reference
  3. Salgia R, Li JL, Lo SH, et al. Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL. J Biol Chem. 1995; 270(10):5039-5047. (Biology). View Reference
  4. Takayama Y, Tanaka S, Nagai K, Okada M. Adenovirus-mediated overexpression of C-terminal Src kinase (Csk) in type I astrocytes interferes with cell spreading and attachment to fibronectin. Correlation with tyrosine phosphorylations of paxillin and FAK. J Biol Chem. 1999; 274(4):2291-2297. (Biology: Immunofluorescence, Immunoprecipitation, Western blot). View Reference
  5. Turner CE, Glenney JR Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990; 111(3):1059-1068. (Biology). View Reference
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610569 Rev. 2

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.