1. What happens if fluid gaps and cracks appear in the resin plug under a prolonged vacuum?
The plugs will re-hydrate rapidly with buffer even after being left to dry under 250 mmHg vacuum for 30 min. IgG capacities should remain unaffected.

2. What happens if air enters the collection tube under vacuum?
Replace the damaged vacuum column barrel first. Always ensure that the threaded hose barb connector has Teflon™ or PTFE tape as the thread sealant. If the problem has not been rectified, replace all the O-rings.

3. Can I use the Midi plug supplied in a spin column kit for the ProVac vacuum assembly?
Unfortunately, the plugs are configured differently for optimal performance under different driving force regimes. We do not recommend switching plugs for centrifugation and vacuum. Only use the plugs in the vacuum kits for the vacuum head.

4. How do I control the vacuum if I have no regulation control on my vacuum pump?
In most circumstances, the vacuum kits will operate under most vacuum pump conditions. If you would like further vacuum control, you will need to purchase a standard 3-way plastic stopcock with a minimum bore size of 4 mm that can accept the appropriate I.D. tubing. As binding capacity is intrinsically linked to flow rate, too high a vacuum will result in lower recoveries of the target antibody. However, under constant vacuum, the flow rate will gradually slow down as increasing sample volumes are loaded on to the Protein A or Protein G column. The upside is that a slower flow rate results in a higher antibody yield.

5. Can I load large volumes of dilute cell culture supernatant with a peristaltic pump?
You can load filtered cell culture supernatant directly into the sample chamber of the vacuum column barrel with a peristaltic pump. Do note that you can use larger collection containers for large volumes of sample. We recommend the following laboratory glass bottles from Fisher Scientific: 100 ml (FB33144) 250 ml (FB33145) 500 ml (FB33146) 1000 ml (FB33147) 2000 ml (FB33148).

6. What should I do if the flow rate slow down during the sample loading step?
In some instances, when high levels of protein are being continuously loaded on to the ProVac plug, we recommend that you disconnect the system from the vacuum pump and then immediately re-apply a weaker vacuum. You only need to remove and then re-attach the vacuum tubing connected to the side-arm of the ProVac manifold. You do not need to disassemble the ProVac system. In this instance, adjust the vacuum to 20 inHg (-10 inHg). If the vacuum pump has no gauge, simply apply a weaker vacuum than that used previously. Higher vacuum pressures can result in the ProVac flow rate slowing down further.