G25脱盐柱使用指南课件

1、Desalting column protocol,Mr. Yu Wang,1,PPT学习交流,Matrix Sephadex G-25 Fine, cross-linked dextran Bed volume 53 ml Bed height 100 mm i.d. 26 mm separation of high (Mr 5 000) from low molecular weight substances (Mr 1 000). Storage +4 to +30 20% ethanol,2,PPT学习交流,长期不用后再使用，应该用5CV平衡柱子 （5*60=300ml buffer）

2、 Try these conditions first Buffer(35ms/cm): 50 mM phosphate buffer, 0.15 M NaCl, pH 7.0 Flow rate: 20 ml/min at room temperature Sample volume: 15 ml Separations: Elute the column with 106 ml (2 CV) of buffer,3,PPT学习交流,Equilibration is not necessary between runs with the same buffer. (去除气泡）Reverse

3、the flow direction and pump 200 ml well de-gassed buffer at a flow rate of 15 ml/min through the column. The quality of the packed bed will not normally be affected.,4,PPT学习交流,5,PPT学习交流,6,PPT学习交流,7,PPT学习交流,Sephadex G-10 is well suited for the separation of biomolecules such as peptides (Mr 700) from

4、 smaller molecules (Mr 30 000 from molecules Mr 1 500 such as labeled protein or DNA from unconjugated dyes. The medium is often used to remove small nucleotides from longer chain nucleic acids. Sephadex G-25 is recommended for the majority of group separations involving globular proteins. These med

5、ia are excellent for removing salt and other small contaminants away from molecules that are greater than Mr 5000.,8,PPT学习交流,sample volumes of up to 30% of the total volume of the desalting column can be processed. not exceed approximately 70 mg/ml。,9,PPT学习交流,Column: HiTrap Desalting, 1 5 ml, 3 5 ml

6、, 5 5 ml Sample: 2 mg/ml BSA in 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0 Sample volume: 28% Vt (1.4, 4.3 and 7.1 ml respectively) Buffer: 50 mM sodium phosphate, 0.15 M sodium chloride, pH 7.0 Flow rate: 5 ml/min,10,PPT学习交流,Storage Store unused media 4C to 30C in 20% ethanol. Do not fre

7、eze. Wash used media with 2 column volumes of distilled water followed by 2 column volumes of 20% ethanol. Store at 4C to 30C. Alternatively, wash with 2 column volumes of distilled water followed by 2 column volumes 0.01 M NaOH. Sodium hydroxide solution is bacteriostatic, easily disposed of and does not shrink the medium. Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back press