To obtain suitable protocol for proteome of cucumber suspension cultured cells,we optimized the pH gradient of IPG strip,the method of protein extraction,reagent and concentration of lysis buffer and SDS-PAGE condition.Results showed pH 4~7 IPG strips,the extraction method acetone precipitation after lysis,lysis buffer containing 8 mol/L urea,2 mol/L thiourea,2% IPG Buffer,4% CHAPS,1% TBP,65 mmol/L DTT,2 mmol/L EDTA,0.001% bromophenol blue and 1% cocktail and 11% gel concentration were appropriate for the sample preparation of cucumber suspension cultured cells.