Jingcheng CrystaroseC FF filler test report
Release time: 2016-08-24
1、Apparatus and consumables
1.1 Instrument: JC-1 automated protein purification system (Jingcheng)
1.2 Chromatographic column: XK16 (GE)
1.3 Jingcheng Biotechnology: CrystaroseC FF(5ml)
1.4 GE:Chelating FF(5ml)
2、Buffer solution preparation
2.1 Equilibrium buffer solution: 20mM Tris-cl,300mM NaCl,PH8.0
2.2 Elution buffer solution 1: 20mM Tris-cl, 300mM NaCl, 50mM Imidazole, PH8.0
2.3 Elution buffer solution 2: 20mM Tris-cl,300mM NaCl,100mM Imidazole,PH8.0
2.4 Elution buffer solution 3: 20mM Tris-cl,300mM NaCl,250mM Imidazole,PH8.0
2.5 Elution buffer solution 4: 20mM Tris-cl,300mM NaCl,500mM Imidazole,PH8.0
3. Operation steps:
3.1.Jingcheng CrystaroseC FF packing test:
3.1.1 Clean the column with deionized water and pour about 5ml of CrystaroseC FF. Equilibrate the chromatographic column with equilibrium buffer solution at a flow rate of 5ml/min.
3.1.2 After the gel is completely settled, draw a horizontal line on the chromatography column with a marker at the horizontal position of the gel.
3.1.3 Adjust the converter on the lower pressure chromatography column so that it is in contact with the packing surface.
3.1.4 Inject the sample with a flow rate of 3 ml/min and a total sample volume of 20 ml.
3.1.5 Rinse the chromatographic column with equilibrium buffer solution until it is close to the baseline parallel.
3.1.6 Elution, sequentially elute with different concentrations of elution buffer, cell phone elution solution.
3.2 GE Chelating FF Test
3.2.1. Pour out all the CrystaroseC FF packing of Jingcheng, disassemble the chromatography column, first rinse the column tube and upper and lower sieves of the column with tap water, and then rinse the column with deionized water. After reassembling the column, pour in GE's Chelating FF packing.
3.2.2. Remove the excess packing, and keep the horizontal position of the upper surface of the packing and the mark line on the surface of the column tube after settling, and equilibrate the chromatographic column with the equilibration buffer solution.
3.2.3. The next steps are the same as 3.1.3 - 3.1.6.
4. Analysis of results:
4.1 By comparing CrystaroseC FF produced by Jingcheng Biotechnology and Chelating FF, a product of GE, it was found that both fillers could obtain higher purity of the target protein after affinity chromatography by metal chelation. However, on the chromatogram, the peak of CrystaroseC FF was late, and a large amount of target protein was eluted by Chelating FF at 100 mM Imidazole, accounting for 65% of the total eluted protein, while the remaining 35% protein was eluted at 250 mM Imidazole and at 50 mM Imidazole. The elution time of CrystaroseC FF was generally late, and only very little of the target protein was eluted at the 10% elution step, and most of the protein was eluted at 250 mM Imidazole, which accounted for 82% of the total eluted protein. At 100 mM and 500 mM Imidazole concentrations a small fraction of the protein was eluted. Therefore, CrystaroseC FF has a higher capacity for protein adsorption and a corresponding increase in retention time. This property can greatly improve the purification performance of the packing material. For example, when purifying some proteins with weak affinity for Ni columns, ordinary Ni columns do not bind or bind very weakly, and they are eluted together with the heteroproteins when washing, making it difficult to obtain effective separation. If the CrystaroseC FF packing is used for purification, it may be effectively adsorbed and purified.
4.2 There is also a difference in protein yield between the two packing materials, the total eluted protein of CrystaroseC FF is 33.9mg, and the total eluted protein of Chelating FF is 29.8mg. The reasons for this are twofold. Firstly, it can be seen from the SDS-PAGE graph that the flow-through solution has a small amount of target protein, and the flow-through protein of Chelating FF is obviously more than that of CrystaroseC FF. Secondly, Chelating FF also lost some of the target proteins in the wash step (Figure 3).
4.3 In terms of chromatogram peak shape, the peak shape of CrystaroseC FF was sharper and the elution peak was better (Figure 1 and Figure 2).
5 Chromatogram
6 SDS-PAGE
Lane 1:CrystaroseC FF flowthrough
Lane 2:Chelating FF flowthrough
Lane 3:CrystaroseC FF elution for 50mM concentration of Imidazole
Lane 4:Chelating FF elution for 50mM concentration of Imidazole
Lane 5:CrystaroseC FF elution for 100mM concentration of Imidazole
Lane 6:Chelating FF elution for 100mM concentration of Imidazole
Lane 7:CrystaroseC FF elution for 250mM concentration of Imidazole
Lane 8:Chelating FF elution for 250mM concentration of Imidazole
Lane 9:CrystaroseC FF elution for 500mM concentration of Imidazole
Lane 10:Chelating FF elution for 500mM concentration of Imidazole
7 Determination of protein concentration
The protein content in the elution was also determined by BCA method, in which the 50mM elution fraction eluted mainly heteroproteins, and it was meaningless to measure the protein concentration, so it was ignored here.
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Jingcheng CrystaroseC FF filler test report
1、Apparatus and consumables 1.1 Instrument: JC-1 automated protein purification system (Jingcheng) 1.2 Chromatographic column: XK16 (GE) 1.3 Jingcheng Biotechnology: CrystaroseC FF(5ml)
Metal chelate chromatography affinity packing comparable to imported products
CrystaroseC FF metal chelate chromatography affinity packing has super high rigidity, higher linear flow rate up to 700cm/h, and the back pressure is close to the imported similar products or even lower. Therefore, the packing life is longer.
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