ABSTRACT
AIM: To extract and purify Syk protein from Sf21 cells transfected by Syk gene. METHODS: Sf21 cells were transfected with recombinant syk gene. After 48 h of incubation at 28 degrees Celsius, the transfected cells were collected and sonicated with Sonfier sonicator on ice. Filtered cell extract was loaded onto a Reactive Yellow-3 resin column and Toyopearl AF-Heparin-650 M column respectively. The characters of Syk protein in the fractions were identified by SDS-PAGE, Western blotting and IEF. RESULTS: 225 mg of protein containing Syk were obtained from Sf21 cells (2.5 x 10(9))extract. There were two subpopulations in the elution of Reactive Yellow-3 resin column with the same relative molecular mass (Mr) 72 x 10(3). The two subpopulations were then applied on Toyopearl AF-Heparin-650 M column and two pure proteins were obtained. The results of SDS-PAGE, Western blotting, and IEF showed the two proteins having the same relative molecular mass (72 x 10(3)), corresponding to Syk, but with different pI. CONCLUSION: The yield of Syk was 8 mg from 2.5 billion cells and the purity was > 95%. The two purified Syk proteins have the same Mr and different pI. The purified Syk protein can be applied to study Syk's mechanism, produce anti-Syk antibody and invent Syk diagnosis kit, etc.
Subject(s)
Intracellular Signaling Peptides and Proteins/isolation & purification , Protein-Tyrosine Kinases/isolation & purification , Spodoptera/enzymology , Animals , Baculoviridae/genetics , Cell Line , Gene Expression , Genetic Vectors , Humans , Intracellular Signaling Peptides and Proteins/genetics , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Spodoptera/cytology , Syk Kinase , TransfectionABSTRACT
AIM: To prepare rabbit anti-Syk antibody and analysis its application in breast cancer diagnosis. METHODS: The rabbits were immunized with recombinant mouse Syk protein to prepare antibody against Syk. The purity and specificity of the antibody were analyzed by Western blot. The expression of Syk protein was detected by immunohistochemical staining. RESULTS: The titer of serum anti-Syk antibody was 1:6 400 (A450 = 1. 03) 6 weeks after the immunization. The SDS-PAGE and Western blot analysis showed that there was a obvious protein band with Mr 72,000, confirming the specificity of obtained anti-Syk antibody. It was displayed by immunohistochemical staining with anti-Sykantibody that in normal breast tissue Syk was high expression; while in infilfrative dustal carcinoma of breast tissue the expression of syk was negative. CONCLUSION: The rabbit anti-Syk antibody with high titer and specificity has been prepared. The Syk is high expression in normal breast tissue, but is deficient in breast cancer tissue. The result may be useful for clinical diagnosis of breast cancer.
