2-Mercaptoethanol → L-Tyrosine

Temperature

4 °C

The transfectants were cultured in RPMI 1640 medium supplemented with 10% FCS (GIBCO), and then centrifuged at 1000 rpm for 5 minutes to recover 2×107 cells. The cell pellets were washed with PBS, dissolved in lysis buffer (20mMTris-HCl, pH7.5, 150 mM NaCl, 2 mM EDTA, Nonidet P-40, 50 mM NaF, 10 mg/ml a protinin, 10 mg/ml leupeptin, 1 mM Na3VO4, 50 mM Na2MoO4, 1 mM phenylmethylsulfonyl fluoride (PMSF)), allowed to stand for 1 hour at 4° C., then centrifuged at 15000 rpm for 30 minutes. Rabbit anti-human FLT3 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to the supernatants and the mixture was stirred for 2 hours at 4° C. After adding Protein A/G Plus agarose (Santa Cruz), the resulting mixture was stirred for 2 hours at 4° C. and washed with the lysis buffer three times. The pellets were then dissolved in sample loading buffer (0.125 M Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 4% SDS), subjected to SDS-PAGE, transferred onto Immobilon PVDF membrane (Millipore), and reacted with anti-phosphorylated tyrosine antibody (4G10, Upstate Biotechnology, Lake Placid, N.Y., USA). Following the reaction, the bands were detected using the ECL system (Amersham). To confirm the presence of FLT3 protein, the same membrane was incubated in stopping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8) for 30 minutes at 70° C., and then reacted with rabbit anti-human FLT3 antibody.

1. customto recover 2×107 cells
2. washThe cell pellets were washed with PBS
3. dissolutiondissolved in lysis buffer (20mMTris-HCl, pH7.5, 150 mM NaCl, 2 mM EDTA, Nonidet P-40, 50 mM NaF, 10 mg/ml a protinin, 10 mg/ml leupeptin, 1 mM Na3VO4, 50 mM Na2MoO4, 1 mM phenylmethylsulfonyl fluoride (PMSF)),
4. waitto stand for 1 hour at 4° C.
5. waitcentrifuged at 15000 rpm for 30 minutes
6. additionRabbit anti-human FLT3 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to the supernatants
7. additionAfter adding Protein A/G Plus agarose (Santa Cruz)
8. stirringthe resulting mixture was stirred for 2 hours at 4° C.
9. washwashed with the lysis buffer three times
10. dissolutionThe pellets were then dissolved in sample loading buffer (0.125 M Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 4% SDS)
11. customreacted with anti-phosphorylated tyrosine antibody (4G10, Upstate Biotechnology, Lake Placid, N.Y., USA)
12. customthe reaction
13. customfor 30 minutes
14. customat 70° C.
15. customreacted with rabbit anti-human FLT3 antibody

ORD record

ord-d61c8c8742c34ca993a6c615bbee1800

Source dataset

uspto-grants-2010_12

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07858333B2