Anti pfak

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-pFAK is a laboratory reagent used to detect and quantify the phosphorylated form of Focal Adhesion Kinase (pFAK) in biological samples. This antibody-based product enables researchers to study the activation and signaling pathways involving pFAK, which is associated with various cellular processes such as cell adhesion, migration, and proliferation.

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Lab products found in correlation

Anti pten, by Merck Group (1 mentions) Dab detection kit, by Roche (1 mentions) Protease inhibitor, by Thermo Fisher Scientific (1 mentions) Anti tubulin, by Merck Group (1 mentions) Anti actin, by Merck Group (1 mentions) Anti neo1, by Santa Cruz Biotechnology (1 mentions) A11012, by Thermo Fisher Scientific (1 mentions) A11001, by Thermo Fisher Scientific (1 mentions) A21042, by Thermo Fisher Scientific (1 mentions) Alexa 488 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Dojindo Laboratories (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Ly294002, by Santa Cruz Biotechnology (1 mentions) Ah7614, by Bio-Techne (1 mentions) Dc260126, by Bio-Techne (1 mentions) A6730, by Merck Group (1 mentions) P erk, by Cell Signaling Technology (1 mentions) Immune blot pvdf membrane, by Bio-Rad (1 mentions) Ecl chemiluminescence method, by Thermo Fisher Scientific (1 mentions) Hrp conjugated anti rabbit or anti mouse abs, by Jackson ImmunoResearch (1 mentions)

Close spelling variants of this product

Rabbit anti-pFAK (2 citations) Anti-pFAK (Y397) (2 citations)

5 protocols using anti pfak

Immunohistochemical Detection of PTEN and p-FAK

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Immunohistochemistry was performed on tissue sections to detect PTEN and p-FAK expression. Samples were incubated with anti-PTEN (mouse monoclonal, clone 6H2.1, Millipore) and anti-p-FAK (rabbit monoclonal, clone 31H5L17, Thermo Fisher) antibodies, followed by DAB Detection Kit (Ventana, Roche). Finally, sections were counterstained with Mayer's hematoxylin.
PTEN and p-FAK positivity was defined by the presence of membranous (p-FAK) and cytoplasmic (PTEN) staining in neoplastic cells. A semiquantitative analysis was performed to provide a score evaluation on a tissue area of a minimum of 6.87 mm² to a maximum of 457.75 mm².
Samples were categorized as: (i) score 0 when no staining was detectable; (ii) score 1 when staining of tumor cells was present in less than 10% of cells; (iii) score 2 when staining involved more than 10% of cells or when weak staining was present in nearly 50% of tumor cells; (iv) score 3 when intense membrane (p-FAK) and cytoplasmic (PTEN) staining was detected in at least 50% of tumor cells.

Cavazzoni A., La Monica S., Alfieri R., Ravelli A., Van Der Steen N., Sciarrillo R., Madeddu D., Lagrasta C.A., Quaini F., Bonelli M., Fumarola C., Cretella D., Digiacomo G., Tiseo M., Peters G.J., Ardizzoni A., Petronini P.G, & Giovannetti E. (2017). Enhanced efficacy of AKT and FAK kinase combined inhibition in squamous cell lung carcinomas with stable reduction in PTEN. Oncotarget, 8(32), 53068-53083.

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Western Blot Protein Extraction and Analysis

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Protein extraction was performed using lysis buffer (SDS 2% w/v, Tris-HCl 80 mM pH 7.5, Glycine 10% w/v) with protease inhibitors (Thermofisher). After three minutes of sonication on ice, samples were centrifuged (10,000 xg) for 5 minutes at 4°C. Samples were resolved in 8% polyacrylamide gels, and proteins were transferred to 0.45 μm nitrocellulose membranes by wet transfer overnight. Primary antibodies were incubated overnight at 4°C in 5% nonfat milk (except for pFAK and NTN1, which is 5% BSA in TBS- 0.01% Tween or RYD systems Buffer 8) diluted in TBS-Tween 0,01%, and the secondary antibodies were incubated at room temperature for 2 h in the same buffer. The antibodies used were anti-NEO1 (# sc-6536, Santa Cruz Biotechnology, 1: 200), anti-NTN1 (AF6419, RYD Systems, 1: 400), anti-actin (A5316, Sigma, 1: 1000), anti-tubulin (T9026, Sigma 1: 1000), anti-pFAK (# 44–624 G, Thermofisher, 1: 1000), total anti-FAK (# 05–537, Millipore 1: 1000), anti-integrin b1 (# sc-8978, Santa Cruz Biotechnology, 1: 300). Western blots were quantified using integrated density analysis with ImageJ software (National Institutes of Health, USA).

Villanueva A.A., Sanchez-Gomez P., Muñoz-Palma E., Puvogel S., Casas B.S., Arriagada C., Peña-Villalobos I., Lois P., Ramírez Orellana M., Lubieniecki F., Casco Claro F., Gallegos I., García-Castro J., Torres V.A, & Palma V. (2021). The Netrin-1-Neogenin-1 signaling axis controls neuroblastoma cell migration via integrin-β1 and focal adhesion kinase activation. Cell Adhesion & Migration, 15(1), 58-73.

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Immunofluorescence Staining of Focal Adhesion Kinase

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The fixation was performed at 37 °C for 15min by 4 % formaldehyde. Cells were permeabilized with PBS containing 0.2 % Triton X-100 (35,501-15, Nacalai Tesque) and 1 % BSA. Cells were stained with anti-pFAK (1:100, 44624G, Thermo Fisher Scientific) and anti-FAK (1:100, 05–537, Merck) antibodies in PBS with 1 % BSA for 1 h at room temperature.
For all staining with secondary antibodies, cells were washed with PBS and stained with the following antibodies in PBS with 1 % BSA: Alexa 488-conjugated goat anti-mouse IgM (1:250, A21042, Thermo Fisher Scientific), Alexa 488-conjugated goat anti-mouse IgG (H + L) (1:300, A11001, Thermo Fisher Scientific), Alexa 594-conjugated goat anti-rabbit IgG (H + L) (1:300, A11012, Thermo Fisher Scientific), or Hoechst 33,342 (1:1000, 346–07951, Dojindo).

Cheng Y.S., Taniguchi Y., Yunoki Y., Masai S., Nogi M., Doi H., Sekiguchi K, & Nakagawa M. (2023). Simultaneous binding of bFGF to both FGFR and integrin maintains properties of primed human induced pluripotent stem cells. Regenerative Therapy, 25, 113-127.

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Investigating Akt and FAK Signaling

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OA sodium salt and A6730 were from Sigma. AG1478 was from Calbiochem. LY294002, Akt2 siRNAs, antibodies (Abs) against Akt1 (5C10), Akt2 (F-7), and FAK (C-20) were from Santa Cruz Biotechnology. Paxillin Ab was from Invitrogen. FFAR4 Ab was from OriGene (Rockville, MD, USA). Phosphospecific Ab to Thr-308/Thr-309 of Akt1/Akt2 (anti-p-Akt-Thr) (244F9), and Ser-473/Ser-474 of Akt1/Akt2 (anti-p-Akt-Ser) (9271S) were from Cell Signaling. Phosphospecific Ab to tyrosine (Tyr)-397 of FAK (anti-p-FAK) was from Invitrogen. Actin monoclonal Ab was kindly provided by PhD Manuel Hernandez (Cinvestav-IPN). DC260126 and AH7614 were from Tocris (Minneapolis, MN, USA). Basement membrane matrix (BD Matrigel) was from BD Biosciences (Bedford, MA, USA). (γ-³²P) ATP was from Perkin-Elmer.

Marcial-Medina C., Ordoñez-Moreno A., Gonzalez-Reyes C., Cortes-Reynosa P, & Perez Salazar E. (2019). Oleic acid induces migration through a FFAR1/4, EGFR and AKT-dependent pathway in breast cancer cells. Endocrine Connections, 8(3), 252-265.

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Western Blot Analysis of Signaling Proteins

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Educated BMMCs were collected by scratching in 10 mL of PBS. After centrifugation, the proteins were isolated using RIPA buffer supplemented with protease and phosphatase inhibitors. 20μL of the obtained lysate were separated by NUPAGE 4-12% Bis-Tris Gel electrophoresis (SDS- PAGE) (Gibco; Amarillo/TX/USA) and transferred to a Immune-Blot PVDF membrane (Biorad; Hercules/Californie/USAs). After transfer, the immune-blots were blocked by incubating with 5% BSA in Tris buffered saline (TBS) (Euromedex; Souffelweyersheim/France) containing 0.05% Tween 20 (VWR; Radnor/Pennsylvanie/USA). The blots were then probed overnight with appropriately diluted primary Abs. After washing in TBS-Tween 0.05%, the membranes were revealed with secondary antibodies for 1 hour at room temperature. After washing, the blots were developed using the ECL chemiluminescence method according to the manufacturer’s protocol (Pierce Chemical; Waltham/MA/USA).
The following antibodies were used to detect their corresponding substrates: anti-pFAK from Invitrogen, FAK from Cell Signalling, pERK from Cell Signaling and anti-tubulin obtained from GeneTex. Either HRP-conjugated anti-rabbit or anti-mouse Abs (Jackson Immunoresearch; Ely/United Kingdom) were used for primary antibody binding.

Bachy S., Wu Z., Gamradt P., Thierry K., Milani P., Chlasta J, & Hennino A. (2022). βig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer. iScience, 25(2), 103758.

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