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IN 45 100 010 • AA • 8 / 12

Optimization of washing and elution

Prolonged wash
A continuously decreasing UV signal during washing is an indication of unbound material being washed out (it
may be target substance if it is weakly bound). Washing should be continued until the UV absorbance signal has
reached 0.01-0.02 (“10-20 mAU”). A BabyBio column should be washed with at least 10 CV buffer.

Optimizing elution conditions
Elution can be carried out using a high salt concentration or by altering the pH to change the charge of the
adsorbed protein. A stronger binding may require higher salt concentration for elution.

Step elution
The optimal salt concentration is dependent on purity and recovery requirements as well as properties of the
target protein and the sample. Using a gradient elution gives increased purity than step elution, but step elution
may be necessary to obtain the highest possible concentration of the target protein. In order to optimize the
salt concentration for step elution an initial gradient test run can be carried out to identify a suitable step elution
conditions for purification of the target protein, see Figure 3.

Note: Remember to take the system dead volume into account when comparing the print out of the gradient
and the trace.

Figure 3. Optimization of step gradient elution with salt. A test run with linear gradient elution gives information about suitable salt concentrations to
be used during the step elution.

Conductivity, gradient elution

UV absorbance

Conductivity, step elution

Volume

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Содержание BabyBio DEAE

Страница 1: ...r basic proteins i e proteins with a high isoelectric point pI while BabyBio Q and BabyBio DEAE columns are suitable for purification of acidic proteins i e proteins with low pI 1 Choose a suitable pH and buffer for the binding of the target protein One pH unit below pI BabyBio S columns or above pI BabyBio Q and BabyBio DEAE columns is a good starting point 2 Connect the column to the chromatogra...

Страница 2: ...therefore interact with an ion exchange resin also at the pI The likelihood of binding to either the cation or the anion exchange resin will increase when moving away from the pI Ion exchange chromatography begins with equilibration of the column to establish the desired pH and charging the resin with suitable counter ions to the charged ligands on the resin e g the negative sulfonate groups can i...

Страница 3: ...f a sample that has not been properly clarified may reduce the performance and lifetime of the column The sample should be applied under conditions similar to those of the binding buffer 2 Connect the column Cut off or twist off the end at the outlet of the column see Figure 2 Note It is of high importance to cut off the tip at the very end of the cone preferable using a scalpel Incorrect removal ...

Страница 4: ...ate may reduce the yield Applied samples should have a pH that gives the target protein a charge that is opposite the charge of the column resin The pH together with the ionic strength in the sample solution might need adjustment for optimal binding 6 Wash After sample application remove unbound impurities by washing the column with 20 30 CV washing buffer or until desired A280 nm absorbance of th...

Страница 5: ...m pressure limit onto the first column If possible the maximum pressure of the chromatography system should be set according to Table 2 Remember always to take the system fluidics contribution to the pressure into account Table 2 Recommended maximum pressure settings for BabyBio columns connected in series Notice that the maximum pressure over each column is always 3 bar No of columns in series Ma...

Страница 6: ...fication using BabyBio S BabyBio Q and BabyBio DEAE Other buffers can possible be used Buffer Product Buffer composition Binding buffer BabyBio S 50 mM Na phosphate pH 7 0 Binding buffer BabyBio Q 50 mM Tris HCl pH 7 4 Binding buffer BabyBio DEAE 50 mM Tris HCl pH 7 4 Elution buffer BabyBio S 50 mM Na phosphate 1 M NaCl pH 7 0 Elution buffer BabyBio Q 50 mM Tris HCl 1 M NaCl pH 7 4 Elution buffer ...

Страница 7: ... jam and thus reduced diffusion rate into the pores A slightly elevated salt concentration reduces but does not eliminate the interactions with the resin by creating a dynamic adsorption desorption equilibrium that allows further diffusion into the resin thus increasing the binding capacity Tuning the flow rate Flow rate is another factor that can be optimized to improve the binding capacity durin...

Страница 8: ...endent on purity and recovery requirements as well as properties of the target protein and the sample Using a gradient elution gives increased purity than step elution but step elution may be necessary to obtain the highest possible concentration of the target protein In order to optimize the salt concentration for step elution an initial gradient test run can be carried out to identify a suitable...

Страница 9: ...as ion exchange chromatography This can be carried out quickly and easily using BabyBio Dsalt 1 ml or 5 ml columns see Related products BabyBio Dsalt columns are also a useful alternative to dialysis for larger sample volumes or when samples need to be processed rapidly to avoid degradation Additional purification Ion exchange chromatography is a powerful single protein purification step or combin...

Страница 10: ...sin using 1 M NaOH applied by a low reversed flow for 2 hours or overnight is often sufficient CIP of the column can be carried out as followed 1 Wash the column with 5 CV deionized water 2 Apply 3 10 CV of 0 5 1 M NaOH for 15 30 minutes Note The contact time is the important factor treatment with NaOH overnight can be necessary if severely fouled 3 Wash the column with 5 10 CV deionized water or ...

Страница 11: ... ml 1 ml 5 ml 1 ml 5 ml Column dimension 7 x 28 mm 1 ml 13 x 38 mm 5 ml 7 x 28 mm 1 ml 13 x 38 mm 5 ml 7 x 28 mm 1 ml 13 x 38 mm 5 ml Recommended flow rate BabyBio 1 ml BabyBio 5 ml 1 ml min 150 cm h 5 ml min 225 cm h 1 ml min 150 cm h 5 ml min 225 cm h 1 ml min 150 cm h 5 ml min 225 cm h Maximum flow rate BabyBio 1 ml BabyBio 5 ml 5 ml min 780 cm h 20 ml min 900 cm h 5 ml min 780 cm h 20 ml min 9...

Страница 12: ... ml 1 x 1 ml 2 x 1 ml 5 x 1 ml 10 x 1 ml 45 200 101 45 200 102 45 200 103 45 200 104 BabyBio S 5 ml 1 x 5 ml 2 x 5 ml 5 x 5 ml 10 x 5 ml 45 200 105 45 200 106 45 200 107 45 200 108 BabyBio Q 1 ml 1 x 1 ml 2 x 1 ml 5 x 1 ml 10 x 1 ml 45 100 101 45 100 102 45 100 103 45 100 104 BabyBio Q 5 ml 1 x 5 ml 2 x 5 ml 5 x 5 ml 10 x 5 ml 45 100 105 45 100 106 45 100 107 45 100 108 BabyBio DEAE 1 ml 1 x 1 ml ...

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