Cells were seeded in 10 cm dishes, left to attach overnight and treated with compounds for 6 h (3 h for FAK-IP) prior to lysis with RIPA buffer. Protein concentration was determined using a BCA assay and 1 mg/mL samples were prepared in a microcentrifuge tube to which 20 μL Dynabeads Magnetic Beads Protein A (Invitrogen, 10002D) were added. 2 μg primary SRC antibody (Cell Signalling, 2109) or appropriate IgG control (Cell Signalling, 2729) was added, and samples rotated at 4 °C overnight. Dynabeads were washed twice with RIPA lysis buffer and three times with PBS before being resuspended in 20 μL of 1X Laemmli sample buffer. Samples were heated to 95°C for 5 min and beads were separated using the magnetic rack before lysates were analyzed for SRC (SCB, SC-8056) and FAK (Cell Signalling, 3285) by standard western blot. For the FAK-IP 1 mg of cell lysate was incubated with anti-FAK (clone 4.47)-conjugated agarose antibody (Millipore, 5-537). The agarose beads were washed twice with RIPA buffer and twice with PBS before being resuspended in 20 μL of 1X Laemmli sample buffer. Samples were heated to 95°C for 5 min and analyzed for SRC (Cell Signalling, 2109) and FAK (Cell Signalling, 3285) by western blot.
Anti fak clone 4.47 conjugated agarose antibody
Manufactured by Merck Group
Sourced in United States
Anti-FAK (clone 4.47)-conjugated agarose antibody is a laboratory reagent used to detect and study Focal Adhesion Kinase (FAK) proteins. It consists of an anti-FAK monoclonal antibody conjugated to agarose beads, allowing for the isolation and purification of FAK proteins from cell lysates or other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads protein a magnetic beads, by Thermo Fisher Scientific (2 mentions)
Anti fak, by Cell Signaling Technology (1 mentions)
Anti psrc y416, by Cell Signaling Technology (1 mentions)
Anti cox 4, by Abcam (1 mentions)
Anti gm130, by BD (1 mentions)
Anti rab7, by Cell Signaling Technology (1 mentions)
Anti paxillin, by BD (1 mentions)
Anti p130cas, by BD (1 mentions)
Anti ifitm3, by Abcam (1 mentions)
Anti pfak y397, by Cell Signaling Technology (1 mentions)
Anti ppaxillin y118, by Cell Signaling Technology (1 mentions)
Anti src clone 36d10, by Cell Signaling Technology (1 mentions)
Phalloidin tritc, by Thermo Fisher Scientific (1 mentions)
Pf562271, by Pfizer (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Dasatinib, by Bristol-Myers Squibb (1 mentions)
3 protocols using anti fak clone 4.47 conjugated agarose antibody
1
Immunoprecipitation of SRC and FAK Proteins
2
Antibody Profiling for Focal Adhesion
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Antibodies used were as follows: anti-Paxillin (RRID:AB_647289 ), anti-GM130 (RRID:AB_398141 ), anti-p130Cas (RRID:AB_397667 ) and anti-RACK1 (RRID:AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID:AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID:AB_10694213 ), anti-FAK (RRID:AB_10694068 ), anti-pFAK Y397 (RRID:AB_2173659 ), anti-pPaxillin Y118 (RRID:AB_2174466 ), anti-Rab7 (RRID:AB_1904103 ), anti-pSrc Y416 (RRID:AB_331697 ), anti-Src (clone 36D10) (RRID:AB_10693939 ), anti-LC3B (RRID:AB_2137707 ) and anti-GAPDH (RRID:AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID:AB_10842517 ), anti-Ambra1 (RRID:AB_2636939 ) and anti-pSrc Y416 (RRID:AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID:AB_310789 ) (Millipore, Billerica, MA, USA). LC3B antibody was from MBL (RRID:AB_1279144 ) (MBL International, Woburn, MA, USA). TRITC-phalloidin was purchased from Life Technologies (RRID:AB_2572408 ) (Paisley, UK). Anti-rabbit (RRID:AB_2099233 ) or mouse (RRID:AB_330924 ) peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb (Princeton, NJ, USA) and PF562271 from Pfizer (Groton, CT, USA).
3
Immunoprecipitation and Western Blot Analysis
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Cells were seeded in 10 cm dishes, left to attach overnight and treated with compounds for 6 hours (3 hours for FAK-IP) prior to lysis with RIPA buffer. Protein concentration was determined using a BCA assay and 1 mg/mL samples were prepared in a microcentrifuge tube, to which, 20 μL Dynabeads Magnetic Beads Protein A (Invitrogen, 10002D) were added. Two micrograms of primary SRC antibody (Cell Signaling Technology, 2109) or appropriate IgG control (Cell Signaling Technology, 2729) was added, and samples rotated at 4°C overnight. Dynabeads were washed twice with RIPA lysis buffer and three times with PBS before being resuspended in 20 μL of 1× Laemmli sample buffer. Samples were heated to 95°C for 5 minutes and beads were separated using the magnetic rack before lysates were analyzed for SRC (SCB, SC-8056) and FAK (Cell Signaling Technology, 3285) by standard Western blot analysis. For the FAK-IP 1 mg of cell lysate was incubated with anti-FAK (clone 4.47)–conjugated agarose antibody (Millipore, 5–537). The agarose beads were washed twice with RIPA buffer and twice with PBS before being resuspended in 20 μL of 1× Laemmli sample buffer. Samples were heated to 95°C for 5 minutes and analyzed for SRC (Cell Signaling Technology, 2109) and FAK (Cell Signaling Technology, 3285) by the Western blot analysis.
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