Abstract: Objective To optimize primary cultures techniques of isolating neonatal rat cardiomyocytes and to compare three different methods of extracting β3-adrenergic receptor（β3-AR）membrane protein from cultured neonatal rat cardiomyocytes. Methods TypeⅡ collagen and differential velocity adhesion were used to collect primary cardiomyocytes. Total protein method, ultracentrifugation method, extract kit method were used to isolate cardiomyocytes β3-AR membrane proteins. The BCA method was applied for protein quantification. Relative content of β3-AR membrane protein and GADPH in the sample were examined by western blot. Results Optimizing culture and isolation skills can produce a great quantity of cardiomyocytes in high concentration.The kit method acquired a higher level of protein concentration（8.26±0.29） g/L than total protein method（5.12±0.47） g/L does than ultracentrifugation method（3.20±0.37） g/L does all of which were with significant difference（P < 0.05） . The concentration of β3-AR membrane protein was higher if obtained by kit method（0.22±0.05）than ultracentrifugation method（0.09±0.03）than total protein method (0.01±0.01) with significant difference（P <0.05） . Conclusion optimizing methodology can obtain abundant myocardial cells in high concentraion. The kit method ofisolating primary cultured β3-AR membrane proteins result in improved concentration and specificity of membrane protein.

Key words: eceptors, adrenergic, beta-3, myocytes, cardiac, extraction, cell culture techniques, cardiomyocytes, β3-
AR membrane protein