Superdex 75 hiload 16 600

You are dissatisfied with the purity of the final preparation of your HRP and decided to include ion-exchange (IEC) and gel-exclusion chromatography (GEC) steps in your purification protocol. From the literature, you found out the following info about HRP enzyme:

Molecular weight: 40,000

Isoelectric point: 7.2

HRP is a very stable protein.

2: Design GEC purification experiment using the information in the appendix. Address and justify the following

I. choice of GEC column

II. choice of the buffer (if you do GEC following the IEC, can you use the same buffer?)

III. where do you expect your protein to elute depending on your chosen column separation range?

IV. suppose HRP tends to form dimers, would your column of choice be able to separate dimers and monomers?

Apendix

Steps in an IEX separation The six steps listed are described in more detail throughout this section. 1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable. 2. Adjust the sample to the chosen starting pHand ionic strength and apply to the column. 3. Wash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, that is when all unbound material has washed through the column. 4. Begin elution using a gradient volume of 10 to 20 CV with an increasing ionic strength up to 500 mM NaCl (50%Bl. Alternatively, elute bound proteins with 5CV of start buffer NaCl at chosen ionic strength. Repeat at higher ionic strengths until the target proteinls) has been eluted. 5. Regeneration: Wash with 5 CV of 1 M Nacl (100%B to elute any remaining ionically bound material 6. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values. 4r Buffer volumes referred to are expressed in column volumes for example 3CV-3 ml for a column with a 1ml bed volume. Using CV to describe a separation profile facilitates method development and transfer of methods to columns of different dimensions. Appendix Figure 5 IAN HiLoad 16/600 Superdex 75 prep grade IBI HILoad 16/600 Superdex 200 prep grode 1. Ferritin IM, 440 000L 0.24 mg/ml 2.IgGIM, 158 000, 02 mg/ml 3 Albumin M, 67000. 5 mg/ml 4. Ovalbumin IM, 43 000.4 mg/ml 5. Myoglobin IM,17000, 1.5 mg/ml Buffer: 50 mM phosphate buffer, 150 mM sodium chloride, 0.01% sodium azide, pH Flow rate: 1.5 ml/min (45 cm/h) 0 o HiLoad 16/600 HiLood 16/600 Superdex prep grade Superdex 200 prepogrode Time min Fig 29. Comparison of the selectivity of Superdex75 prep grade and Superde 200 prep grade for standard proteins. Superdex 75 prep grade Algivesexcelent resolution of the three protensin the molecular weightrange of 17 000 to67 000 while the two longest proteins ferritinand gG, peoksland21elute together in the void volume M Superdex 200 prep grode IBI resolves the two largest proteins completely, however. The ferritinlpeak 1lcontains aggregates that resultin the observed double peak.

Steps in an IEX separation The six steps listed are described in more detail throughout this section. 1. Equilibrate column with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable. 2. Adjust the sample to the chosen starting pHand ionic strength and apply to the column. 3. Wash with 5 to 10 CV of start buffer or until the baseline, eluent pH, and conductivity are stable, that is when all unbound material has washed through the column. 4. Begin elution using a gradient volume of 10 to 20 CV with an increasing ionic strength up to 500 mM NaCl (50%Bl. Alternatively, elute bound proteins with 5CV of start buffer NaCl at chosen ionic strength. Repeat at higher ionic strengths until the target proteinls) has been eluted. 5. Regeneration: Wash with 5 CV of 1 M Nacl (100%B to elute any remaining ionically bound material 6. Re-equilibrate with 5 to 10 CV of start buffer or until eluent pH and conductivity reach the required values. 4r Buffer volumes referred to are expressed in column volumes for example 3CV-3 ml for a column with a 1ml bed volume. Using CV to describe a separation profile facilitates method development and transfer of methods to columns of different dimensions. Appendix Figure 5 IAN HiLoad 16/600 Superdex 75 prep grade IBI HILoad 16/600 Superdex 200 prep grode 1. Ferritin IM, 440 000L 0.24 mg/ml 2.IgGIM, 158 000, 02 mg/ml 3 Albumin M, 67000. 5 mg/ml 4. Ovalbumin IM, 43 000.4 mg/ml 5. Myoglobin IM,17000, 1.5 mg/ml Buffer: 50 mM phosphate buffer, 150 mM sodium chloride, 0.01% sodium azide, pH Flow rate: 1.5 ml/min (45 cm/h) 0 o HiLoad 16/600 HiLood 16/600 Superdex prep grade Superdex 200 prepogrode Time min Fig 29. Comparison of the selectivity of Superdex75 prep grade and Superde 200 prep grade for standard proteins. Superdex 75 prep grade Algivesexcelent resolution of the three protensin the molecular weightrange of 17 000 to67 000 while the two longest proteins ferritinand gG, peoksland21elute together in the void volume M Superdex 200 prep grode IBI resolves the two largest proteins completely, however. The ferritinlpeak 1lcontains aggregates that resultin the observed double peak.