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Total fak

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Total FAK is a laboratory equipment designed to measure focal adhesion kinase (FAK) activity. It provides a quantitative analysis of FAK levels in cell samples.

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Lab products found in correlation

P mlc2, by Cell Signaling Technology (4 mentions) Tenascin c, by Abcam (4 mentions) Py397 fak, by Thermo Fisher Scientific (4 mentions) Anti α tubulin, by Santa Cruz Biotechnology (4 mentions) Fak s910, by Thermo Fisher Scientific (4 mentions) Anti rab40c, by Santa Cruz Biotechnology (4 mentions) Anti ha, by Santa Cruz Biotechnology (4 mentions) Fibronectin, by Abcam (2 mentions) Gli 1, by Thermo Fisher Scientific (2 mentions) Collagen 12a1, by Santa Cruz Biotechnology (2 mentions) Integrin β1, by Merck Group (2 mentions) Mabt409, by Thermo Fisher Scientific (2 mentions) P mypt1, by Merck Group (2 mentions) Collagen 3, by Abcam (2 mentions) Total stat3, by Cell Signaling Technology (2 mentions) Rock1, by Cell Signaling Technology (2 mentions) Rock2, by Cell Signaling Technology (2 mentions) P stat3, by Cell Signaling Technology (2 mentions) P smad2 3, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions)

12 protocols using total fak

1

Immunohistochemistry Antibody Panel

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Antibodies were as follows: a-SMA (Sigma-Aldrich C6198, 1:1000), GLi-1 (Thermo Scientific PA5-32206, 1:500), Tenascin C (Abcam AB108930, 1:500), Fibronectin (Abcam AB2413, 1:500), Collagen 12A1 (Santa Cruz Biotechnology E-15 sc-68449, 1:200), Sox2 (Abcam ab97959, 1:500), Vimentin (Cell Signaling 5741S, 1:200), FAP (Abcam AB53066, 1:500), Collagen III (Abcam AB7778, 1:500), YAP (Cell signaling 4912, 1:200), β1integrin (EMD Millipore MABT409, 1:500), pY397-FAK (Invitrogen 44625, 1:200), total FAK (BD Biosciences 610088, 1:1,000), ROCK1 (Cell Signaling C8F7, 1:1,000), ROCK2 (Cell Signaling D1B1, 1:1,000), p-MLC2 (Cell Signaling 3671, 1:200), p-MyPT1 (Millipore ABS45, 1:200), p-Stat3 (Cell Signaling 9145, 1:200), p-SMAD2/3 (Cell Signaling 8828, 1:200), total Stat3 (Cell Signaling 9132, 1:1,000), GAPDH (Cell Signaling 2118, 1:5,000), CD45 (BD Biosciences 550539, 1:200), CD68 (Thermo Scientific Ab-3, 1:200), Alexa Fluor-conjugated goat secondary anti–mouse IgG and anti–rabbit IgG antibodies (Invitrogen A11012, 1:1,000 and A11005, 1:1,000) and HRP-conjugated rabbit secondary antibody (GE health care life sciences NA934VS, 1:5,000).
Laklai H., Miroshnikova Y.A., Pickup M.W., Collisson E.A., Kim G.E., Barrett A.S., Hill R.C., Lakins J.N., Schlaepfer D.D., Mouw J.K., LeBleu V.S., Roy N., Novitskiy S.V., Johansen J.S., Poli V., Kalluri R., Iacobuzio-Donahue C.A., Wood L.D., Hebrok M., Hansen K., Moses H.L, & Weaver V.M. (2016). Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression. Nature medicine, 22(5), 497-505.
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2

Antibody Validation for Cellular Analysis

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Antibodies were as follows (used at 1μg ml−1 for western blotting and chromatin immunoprecipitation (ChIP) and at indicated concentrations for immunofluorescence studies): tenascin C (Abcam ab108930; 1:1,000), pY397-FAK (Invitrogen 44625; 1:200), total FAK (BD Biosciences 610088), pMLC2 (Cell Signaling 3671; 1:200), total MLC2 (Abcam ab92721, clone EPR3741; 1:200), p-MyPT1 (Millipore ABS45; 1:200), hyaluronic acid binding protein (Calbiochem, 385911; 1:500), aggrecan (Abcam ab3778; 1:500), versican (Abcam ab19345; 1:500), collagen 1 (Abcam ab34710; 1:1,000), propidium iodide (AcrosOrganics 440300250; 1 μg ml−1), β-actin (Sigma-Aldrich a5441), HIF1α (Abcam ab-1, for immunofluorescence; 1:200; Abcam ab1, for ChIP; Novus 100-449, for western blotting), hypoxyprobe (Hypoxyprobe, HP1-100 Kit; per manual), CD31 (BD 550389; 1:500), laminin (Abcam ab11575; 1:500), RNA polymerase II (Millipore 05-623B), rabbit IgG isotype control (Cell Signaling 2729; 1:500), Alexa Fluor-conjugated goat secondary anti-mouse IgG and anti-rabbit IgG antibodies (Invitrogen A11012 and A11005; 1:500) and HRP-conjugated rabbit secondary antibody (GE Healthcare Life Sciences NA934VS; 1:5,000).
Miroshnikova Y.A., Mouw J.K., Barnes J.M., Pickup M.W., Lakins J.N., Kim Y., Lobo K., Persson A.I., Reis G.F., McKnight T.R., Holland E.C., Phillips J.J, & Weaver V.M. (2016). Tissue mechanics promote IDH1-dependent HIF1α–tenascin C feedback to regulate glioblastoma aggression. Nature cell biology, 18(12), 1336-1345.
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3

Antibody Verification and Characterization

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The following antibodies were used in this study: anti-FLAG (clone M2, WB 1:1,000; Sigma-Aldrich), anti-GAPDH (WB 1:5,000; UBPBio), FAK S910 (WB 1:1,000, 44-596G; Invitrogen), total FAK (WB 1:1,000, 610087; BD Biosciences), and paxillin (IF 1:500; Transduction Labs). Anti-HA (WB 1:500, IP 2 μg/1 mg cell lysate, SC F-7), anti–α-tubulin (WB 1:5,000, 23948), anti-Rab40c (WB 1:500, H-8 sc514826), cul-5 (WB 1:500, H-300), and mouse ANKRD28 (WB 1:500) were purchased from Santa Cruz Biotechnology. MOB1(E1N9D) and p-MOB1(D2F10) were purchased from Cell Signaling Technology. Rabbit anti-SAPS1/2/3, ANKRD28/52, and PP6c were purchased from Bethyl Laboratories. Specificity of anti-Rab40c antibody was confirmed by immunoblotting lysates derived from cells expressing FLAG-Rab40a, FLAG-Rab40b, or FLAG-Rab40c constructs (Fig S2A).
Han K.J., Mikalayeva V., Gerber S.A., Kettenbach A.N., Skeberdis V.A, & Prekeris R. (2022). Rab40c regulates focal adhesions and PP6 activity by controlling ANKRD28 ubiquitylation. Life Science Alliance, 5(9), e202101346.
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4

Quantification of FAK Phosphorylation

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Cells were adhered to collagen-coated dishes in keratinocyte growth medium as described in the Derivation of MK cell variants section, under which conditions they deposit endogenous LN-332 (Choma et al., 2004 (link)). Total lysates were prepared in cell lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitors, and protein concentrations were determined using the BCA Protein Assay kit (Thermo Fisher Scientific). Equal protein was resolved by nonreducing 10% SDS/PAGE, transferred to nitrocellulose, and probed with the antibodies anti-FAK pY397 (Cell Signaling Technology), anti-FAK pY861 (Abcam), anti-FAK pY925 (Cell Signaling Technology), or total FAK (BD). Chemiluminescence was performed using the SuperSignal kit (Thermo Fisher Scientific) and then was quantified using a ChemiDoc MP imaging system with Image Lab software (Bio-Rad Laboratories).
Longmate W.M., Lyons S.P., Chittur S.V., Pumiglia K.M., Van De Water L, & DiPersio C.M. (2017). Suppression of integrin α3β1 by α9β1 in the epidermis controls the paracrine resolution of wound angiogenesis. The Journal of Cell Biology, 216(5), 1473-1488.
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5

Western Blotting Analysis of Signaling Proteins

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Western blotting was performed as previously described (36 ) using the following antibodies: total FAK (BD Bioscience:610087), total p130Cas (BD Bioscience:610271); pY397FAK (Abcam, Cambridge, MA:ab81298); pY416Src (Cell Signaling, Danvers, MA:2101), total Src (Cell Signaling:2109), pY410p130Cas (Cell Signaling:4011), total c-Jun (Cell Signaling:2315), pS63c-Jun (Cell Signaling:2361); pY861FAK (Invitrogen, Carlsbad, CA:44–626G); α-tubulin (CALBIOCHEM, Burlington, MA:CP06). Phosphorylated protein expression was normalized to total protein for quantification using the Odyssey CLx imager (Li-Cor).
Kessler B.E., Mishall K.M., Kellett M.D., Clark E.G., Pugazhenthi U., Pozdeyev N., Kim J., Tan A.C, & Schweppe R.E. (2018). Resistance to Src inhibition alters the BRAF-mutant tumor secretome to promote an invasive phenotype and therapeutic escape through a FAK>p130Cas>c-Jun signaling axis. Oncogene, 38(14), 2565-2579.
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6

Immunohistochemistry Antibody Panel

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Antibodies were as follows: a-SMA (Sigma-Aldrich C6198, 1:1000), GLi-1 (Thermo Scientific PA5-32206, 1:500), Tenascin C (Abcam AB108930, 1:500), Fibronectin (Abcam AB2413, 1:500), Collagen 12A1 (Santa Cruz Biotechnology E-15 sc-68449, 1:200), Sox2 (Abcam ab97959, 1:500), Vimentin (Cell Signaling 5741S, 1:200), FAP (Abcam AB53066, 1:500), Collagen III (Abcam AB7778, 1:500), YAP (Cell signaling 4912, 1:200), β1integrin (EMD Millipore MABT409, 1:500), pY397-FAK (Invitrogen 44625, 1:200), total FAK (BD Biosciences 610088, 1:1,000), ROCK1 (Cell Signaling C8F7, 1:1,000), ROCK2 (Cell Signaling D1B1, 1:1,000), p-MLC2 (Cell Signaling 3671, 1:200), p-MyPT1 (Millipore ABS45, 1:200), p-Stat3 (Cell Signaling 9145, 1:200), p-SMAD2/3 (Cell Signaling 8828, 1:200), total Stat3 (Cell Signaling 9132, 1:1,000), GAPDH (Cell Signaling 2118, 1:5,000), CD45 (BD Biosciences 550539, 1:200), CD68 (Thermo Scientific Ab-3, 1:200), Alexa Fluor-conjugated goat secondary anti–mouse IgG and anti–rabbit IgG antibodies (Invitrogen A11012, 1:1,000 and A11005, 1:1,000) and HRP-conjugated rabbit secondary antibody (GE health care life sciences NA934VS, 1:5,000).
Laklai H., Miroshnikova Y.A., Pickup M.W., Collisson E.A., Kim G.E., Barrett A.S., Hill R.C., Lakins J.N., Schlaepfer D.D., Mouw J.K., LeBleu V.S., Roy N., Novitskiy S.V., Johansen J.S., Poli V., Kalluri R., Iacobuzio-Donahue C.A., Wood L.D., Hebrok M., Hansen K., Moses H.L, & Weaver V.M. (2016). Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression. Nature medicine, 22(5), 497-505.
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7

Western Blotting Protein Expression Analysis

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Western blotting was performed as previously described (36 ) using the following antibodies: total FAK (BD Bioscience:610087), total p130Cas (BD Bioscience:610271); pY397FAK (Abcam, Cambridge, MA:ab81298); pY416Src (Cell Signaling, Danvers, MA:2101), total Src (Cell Signaling:2109), pY410p130Cas (Cell Signaling:4011), total c-Jun (Cell Signaling:2315), pS63c-Jun (Cell Signaling:2361); pY861FAK (Invitrogen, Carlsbad, CA:44–626G); α-tubulin (CALBIOCHEM, Burlington, MA:CP06). Phosphorylated protein expression was normalized to total protein for quantification using the Odyssey CLx imager (Li-Cor).
Kessler B.E., Mishall K.M., Kellett M.D., Clark E.G., Pugazhenthi U., Pozdeyev N., Kim J., Tan A.C, & Schweppe R.E. (2018). Resistance to Src inhibition alters the BRAF-mutant tumor secretome to promote an invasive phenotype and therapeutic escape through a FAK>p130Cas>c-Jun signaling axis. Oncogene, 38(14), 2565-2579.
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8

Antibody Validation for Cell Analysis

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The following antibodies were used in this study: anti-FLAG (WB 1:1,000, F3165; Sigma), anti-EPLIN (WB 1:1,000, IF 1:100, 50311; Cell Signaling Technology), anti-EPLIN (WB 1:1,000, IF 1:100, sc-136399; Santa Cruz Biotechnology), anti-EPLIN (WB 1:1,000, immunoprecipitation [IP] 1 μg/1 mg lysate, immunohistochemistry [IHC] 1:200, 16639–1-AP; Proteintech), anti-HA (WB 1:500, IP 2 µg/1 mg cell lysate, SC F-7; Santa Cruz Biotechnology), Alexa Fluor 568–phalloidin (IF 1:1,000, A1238; Life Technologies), SiR-actin (1:10,000, CY-SC001; Cytoskeleton), anti–β-tubulin (WB 1:2,500, 926–42211; LI-COR), anti–α-tubulin (WB 1:2,500, 23948; Santa Cruz Biotechnology), pFAK Y397 (WB 1:1,000, ab8129; Abcam), FAK S910 (WB 1:1,000, 44-596G; Invitrogen), anti–E-Cadherin (WB 1:1,000, IHC 1:200, 3195; Cell Signaling Technology), total FAK (WB 1:1,000, 610087; BD Biosciences), anti-zyxin (IF 1:1,000, ab50391; Abcam), anti-Rab40b (WB 1:500, LS-C353287; LSBio), anti-Rab40c (WB 1:500, H-8 sc-514826; Santa Cruz Biotechnology), and anti-CD63 (IF 1:100; gift from Andrew Peden, University of Shefield, Shefield, UK).
Linklater E.S., Duncan E.D., Han K.J., Kaupinis A., Valius M., Lyons T.R, & Prekeris R. (2021). Rab40–Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration. The Journal of Cell Biology, 220(7), e202008060.
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9

Antibody Validation for Cellular Analysis

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Antibodies were as follows (used at 1μg ml−1 for western blotting and chromatin immunoprecipitation (ChIP) and at indicated concentrations for immunofluorescence studies): tenascin C (Abcam ab108930; 1:1,000), pY397-FAK (Invitrogen 44625; 1:200), total FAK (BD Biosciences 610088), pMLC2 (Cell Signaling 3671; 1:200), total MLC2 (Abcam ab92721, clone EPR3741; 1:200), p-MyPT1 (Millipore ABS45; 1:200), hyaluronic acid binding protein (Calbiochem, 385911; 1:500), aggrecan (Abcam ab3778; 1:500), versican (Abcam ab19345; 1:500), collagen 1 (Abcam ab34710; 1:1,000), propidium iodide (AcrosOrganics 440300250; 1 μg ml−1), β-actin (Sigma-Aldrich a5441), HIF1α (Abcam ab-1, for immunofluorescence; 1:200; Abcam ab1, for ChIP; Novus 100-449, for western blotting), hypoxyprobe (Hypoxyprobe, HP1-100 Kit; per manual), CD31 (BD 550389; 1:500), laminin (Abcam ab11575; 1:500), RNA polymerase II (Millipore 05-623B), rabbit IgG isotype control (Cell Signaling 2729; 1:500), Alexa Fluor-conjugated goat secondary anti-mouse IgG and anti-rabbit IgG antibodies (Invitrogen A11012 and A11005; 1:500) and HRP-conjugated rabbit secondary antibody (GE Healthcare Life Sciences NA934VS; 1:5,000).
Miroshnikova Y.A., Mouw J.K., Barnes J.M., Pickup M.W., Lakins J.N., Kim Y., Lobo K., Persson A.I., Reis G.F., McKnight T.R., Holland E.C., Phillips J.J, & Weaver V.M. (2016). Tissue mechanics promote IDH1-dependent HIF1α–tenascin C feedback to regulate glioblastoma aggression. Nature cell biology, 18(12), 1336-1345.
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10

Antibodies for Cellular Imaging and Signaling

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Antibodies used in this study are as follows: anti-Flag -(WB 1:1000, Sigma F3165), anti-EPLIN -(WB 1:1000, IF 1:100, Cell Signaling Technology 50311) , anti-EPLIN (WB 1:1000, IF 1:100, Sant Cruz Biotechnology sc-136399), anti-EPLIN (WB 1:1000, IP 1 ug/1 mg lysate, IHC 1:200 Proteintech 16639-1-AP), anti-HA (WB 1:500, IP 2 μg/1mg cell lysate, Santa Cruz SC F-7) , Alexa-Fluor-568-phalloidin (IF 1:1000, Life Technologies A1238), SiR-Actin -(1:10,000, Cytoskeleton CY-SC001), anti-β-tubulin -(WB 1:2500, LiCor 926-42211), anti-α-tubulin -(WB 1:2500, Santa Cruz 23948), pFAK Y397 (WB 1:1000, Abcam ab8129), FAK S910 (WB 1:1000, Invitrogen 44-596G), anti-E-Cadherin (WB 1:1000, IHC 1:200, Cell Signaling 3195), total FAK (WB 1:1000, BD Biosciences 610087), anti-Zyxin (IF 1:1000 abcam, ab50391), anti-Rab40b (WB 1:500, LSBio, LS-C353287), anti-Rab40c (WB 1:500 Santa Cruz, H-8 sc-514826), anti-CD63 (IF 1:100, gift from Dr. Andrew Peden).
Linklater E.S., Duncan E.D., Han K., Kaupinis A., Valius M., Lyons T.R., & Prekeris R. (2021). Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion.
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