Xiaojing.Wang at 15:41, 1 March 2012

2012-03-01T15:41:26Z

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== Step Gradient ==

Concentration of lentiviral by anion exchange chromatography using '''Mustang Q anion exchange membrane''' or '''Sartobind Q 75X  '''cartridges

1. Set up two Acrodisk Mustang Q membrane disks.

1. Add 25 ml of PBS to 50 ml BD or VWR/Fisher tubes.

2. Add 20 ml of '''Buffer C '''to 204 ml of the lentivirus-containing cell culture supernatant

3. Load 32 ml of supernatant (step 2) onto the tandem Acrodisk units using a syringe. Avoid the formation of bubbles during loading of the sample.

4. Wash Acrodisk units with 15 ml of '''Buffer A''' using a syringe. Avoid the formation of bubbles.

5. Elute lentivirus with 3 ml of '''Buffer B''' using a syringe. Collect the eluate in a 50 ml tube, add water (10 vol) to dilute to 0.15M NaCl.

6. Elute lentivirus with 3 ml of'''  1:1 mixture of Buffer C + Buffer A ''' using a syringe. Collect the eluate in a 50 ml tube, add water (10 vol) to dilute to 0.15M NaCl. 

6. Regenerate Acrodisks by passing 5 ml of a 1 N NaOH solution through followed by 5 ml of '''Buffer A'''.

7. Load another 32 ml of supernatant (Step 2) and proceed with Step 6, repeat this cycle four more times until all of the supernatant has been processed.

8. Concentrate lentivirus using 100 KDa spin concentrator, use PBS to exchange buffer.

9. Aliquot lentivirus into screw-cap microfuge tubes in in 25- to 100-ul portions.

10. Snap-freeze tubes in crushed dry ice and store at -80 oC.

 

'''Buffer A'''

0.15 M NaCl, 25 mM Tris-HCl, pH 8.0 or pH 7.5;

'''Buffer B''' <strike>400</strike> 300 mM NaCl, 25 mM Tris-HCl, pH 8.0 or pH 7.5;

'''Buffer C''' 1.5 M NaCl, 25 mM Tris-HCl, pH 8.0 or pH 7.5. 

 

== 10 ml ==

1. Set up two Acrodisk Mustang Q membrane disks.

2. Add 1 ml of '''Buffer C''' to 10 ml of the lentivirus-containing cell culture supernatant

3. Load 11 ml of supernatant (step 2) onto the tandem Acrodisk units using a syringe. Avoid the formation of bubbles during loading of the sample.

4. Wash Acrodisk units with 15 ml of '''Buffer A''' using a syringe. Avoid the formation of bubbles.

5. Elute lentivirus with 3 ml of '''Buffer B''' using a syringe. Collect the eluate in a 50 ml tube, add water (3 ml) to dilute to 0.15M NaCl.

6. Elute lentivirus with 3 ml of '''1:1 mixture of Buffer C + Buffer A''' using a syringe. Collect the eluate in a 50 ml tube, add water (13.5 ml) to dilute to 0.15M NaCl.

6. Regenerate Acrodisks by passing 5 ml of a 1 N NaOH solution through followed by 5 ml of '''Buffer A'''.

7. Concentrate lentivirus using 100 KDa spin concentrator, use PBS to exchange buffer.

8. Aliquot lentivirus into screw-cap microfuge tubes in 25- to 100-ul portions.

9. Snap-freeze tubes in crushed dry ice and store at -80 oC. 

 

== FPLC ==

 1. Adjust  supernatant (500 ml)  to 25 mM Tris-HCl, pH 8.0, 0.3 M NaCl.

2. Load onto a Mustang Q Acrodisc (bed volume 0.18 ml) at a flow rate of 10 ml/min for 8 min

3. Wash with 2 ml of 25 mM Tris-HCl, pH 8.0, 0.3 M NaCl.

4. Elute with a 10-ml gradient ranging from 0.3 to 1.5 M NaCl in 25 mM Tris-HCl,pH 8.0, collect 1-ml fractions. 

[[Category:Protocols]]

Lentivirus Concentration Q Membrane - Revision history

Xiaojing.Wang at 15:41, 1 March 2012