The invention discloses a
western blot detection method for
phosphoprotein. The
western blot detection method for the
phosphoprotein comprises the following steps: S1, preparing a sample, carrying outgel
electrophoresis, namely adding 6 times SDS sample loading
buffer solution in a ratio of 5:1 into the
phosphoprotein sample, uniformly mixing, and carrying out
electrophoresis at a
voltage of 80 V; S2, transferring a membrane, namely connecting a gel surface with a negative pole, connecting a PVDF membrane with a positive pole, and carrying out
transfer printing for 1 hour at a
constant voltage of 130 V; S3, sealing, namely putting the PVDF membrane into PBST containing 5% of BSA, and
hatching for 20 minutes in a shaking table and at
room temperature; S4, adding primary antibodies, namelyadding the primary antibodies,
hatching for 2 hours in the shaking table and at the temperature of 24 DEG C, and rinsing for four times with PBST, wherein the rinsing is carried out once every 5 minutes; S5, adding secondary antibodies, namely adding anti-rabbit secondary antibodies labelled by
horse radish peroxidase (HRP),
hatching for 1 hour in the shaking table and at
room temperature, and rinsing for four times with PBST, wherein the rinsing is carried out once every 5 minutes; and S6, developing, namely uniformly dropwise adding a developing
reagent on the PVDF membrane, and carrying outgel developing. The
western blot detection method for the phosphoprotein has the advantages that a wet membrane transferring manner is adopted during western
blot detection of the phosphoprotein, anda
protein signal is continuous and high in strength; and no non-specific band exists when
dilution ratio of the primary antibodies is 1:5000, and the
protein signal is better in effect.