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| | In Vitro Activity Assay |
| Description: | The in vitro activity of the compounds of the compounds of the invention may be determined by the following procedure. More particularly, the following assay provides a method to determine whether compounds of the compounds of the invention inhibit the tyrosine kinase activity of the catalytic construct FAK(410-689). The assay is an ELISA-based format, measuring the inhibition of poly-glu-tyr phosphorylation by FAK(410-689). The assay protocol has three parts:I. Purification and cleavage of His-FAK(410-689)II. FAK410-689 (a.k.a. FAKcd) ActivationIII. FAKcd Kinase ELISAMaterials: Ni-NTA agarose (Qiagen) XK-16 column (Amersham-Pharmacia) 300 mM Imidizole Superdex 200 HiLoad 16/60 prep grade column (Amersham Biotech.) Antibody: Anti-Phosphotyrosine HRP-Conjugated Py20 (Transduction labs) FAKcd: Purified and activated in house TMB Microwell Peroxidase Substrate (Oncogene Research Products #CL07) BSA: Sigma #A3294 Tween-20: Sigma #P1379 DMSO: Sigma #D-5879 D-PBS: Gibco #14190-037 Reaqents for Purification: Buffer A: 50 mM HEPES pH 7.0 500 mM NaCl 0.1 mM TCEP Complete protease inhibitor cocktail tablets (Roche) Buffer B: 25 mM HEPES pH 7.0 400 mM NaCl 0.1 mM TCEP Buffer C: 10 mM HEPES pH 7.5 200 mM Ammonium Sulfate 0.1 mM TCEP Reagents for Activation: FAK(410-689): 3 tubes of frozen aliquots at 150 μl/tube for a total of 450 μl at 1.48 mg/ml (660 μg) His-Src(249-524): 0.74 mg/ml stock in 10 mM HEPES, 200 mM (NH4)2SO4 Src reaction buffer (Upstate Biotech): 100 mM Tris-HCl pH7.2 125 mM MgCl2 25 mM MnCl2 2 mM EDTA 250 μM Na3VO4 2 mM DTT Mn2+/ATP cocktail (Upstate Biotech) 75 mM MnCl2 500 μM ATP 20 mM MOPS pH 7.2 1 mM Na3VO4 25 mM glycerol phosphate 5 mM EGTA 1 mM DTT ATP: 150 mM stock MgCl2: 1 M Stock DTT: 1 M stock Reagents for FAKcd Kinase ELISA: Phosphorylation Buffer: 50 mM HEPES, pH 7.5 125 mM NaCl 48 mM MgCl2 Wash Buffer: TBS+0.1% Tween-20. Blocking Buffer: Tris Buffer Saline 3% BSA 0.05% Tween-20, filtered Plate Coating Buffer: 50 mg/ml Poly-Glu-Tyr (Sigma #P0275) in Phosphate buffer Saline (DPBS). ATP: 0.1M ATP in H2O or HEPES, pH7 Note: ATP Assay Buffer: Make up as 75 uM ATP in PBS, so that 80 μl in 120 μl reaction volume=50 μM final ATP concentration. I. Purification of His-FAKcd(410-689): 1. Resuspend 130 g baculovirus cell paste containing the over expressed His-FAKcd410-689 recombinant protein in 3 volumes (400 ml) of Buffer A. 2. Lyse cells with one pass on a microfluidizer. 3. Remove cell debris by centrifugation at 4° C. for 35 minutes at 14,000 rpm in a Sorval SLA-1500 rotor. 4. Transfer the supernatant to a clean tube and add 6.0 ml of Ni-NTA agarose (Qiagen). 5. Incubate the suspension with gentle rocking at 4° C. for 1 hour. 6. Centrifuge suspension at 700×g in a swinging bucket rotor. 7. Discard the supernatant and resuspend the agarose beads in 20.0 ml of Buffer A. 8. Transfer the beads to an XK-16 column (Amersham-Pharmacia) connected to a FPLC. 9. Wash the agarose-beads with 5 column volumes of Buffer A and elute off the column with a step gradient of Buffer A containing 300 mM Imidizole. 10. Perform a buffer exchange of the eluted fractions into Buffer B. 11. Following buffer exchange, pool the fractions and add thrombin at a 1:300 (w/w) ratio and incubated overnight at 13° C. to remove the N-terminal His-tag (His-FAK410-698 FAK410-689 (a.k.a. FAKcd)). 12. Add the reaction mixture back onto the Ni-NTA column equilibrated with Buffer A and collect the flow-through. |
| Affinity data for this assay | |
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