1. Q: What are PhyTip columns?
A: PhyTip columns are part of a unique protein purification and enrichment system offered by PhyNexus. The complete system includes the instrument platform to automate the process of Capture, Purify and Enrich plus the PhyTip columns. The PhyTip columns are novel devices containing specific affinity resins, e.g. Protein A that are maintained at the base of a pipette tip and used to capture the protein of interest by bidirectional flow of sample volume.
2. Q: Why would I use PhyTip columns?
A: PhyTip columns have been optimized to produce the highest possible purity and either yield or concentration of sample proteins. They have also been optimized to be able to perform these functions with low sample volumes, thus saving valuable resources and enabling optimized high throughput expression of protein samples. The columns are disposable and can operate rapidly to perform a purification in 15 minutes in a high throughput, walk-away manner.
3. Q: What are PhyTip columns made of?
A: All PhyTip columns are manufactured from polypropylene and polyester polymers and resin beds.
4. Q: What is the mesh made of?
A: The mesh retainer is a hydrophilic, bio compatible polymer.
5. Q: Is the PhyTip insert removable?
A: No, it has been designed and manufactured to remain in place.
6. Q: Are PhyTip columns sterile?
A: No. PhyTip columns are shipped in glycerol which is a bacterialside. The columns may be stored at room temperature prior to processing for several hours with no degradation, but PhyTip columns are not sterile.
7. Q: How are PhyTip columns QC tested?
A: PhyTip columns are tested for backpressure to ensure reproducible flow rates. Follow the recommended procedures found in the manual and instrument software for using PhyTip columns.
8. Q: Why are PhyTip columns shipped with some fluid in them, and what is that fluid?
A: PhyTip columns are delivered with glycerol shipping fluid in order to keep the resin bed moist which is necessary for optimal performance. The PhyTip affinity columns may be used straight out of the box without preconditioning. The first step of processing is a conditioning step. The PhyTip gel filtration columns do need to be conditioned overnight prior to use by adding water to the tip box and to the top of each column. Check the specification sheet for details on the resin-specific shipping liquid.
9. Q: How clear or clean should my sample be before I begin to use a PhyTip column?
A: PhyTip columns have been used with a variety of different starting materials, e.g. periplasmic extracts, lysates etc. In all cases we recommend that the solution to be purified is clear of any particulate substances. In order to guarantee this, make sure that the sample to be purified has been spun in a centrifuge so that only the clear supernatant is used. Care should be taken not to disturb the plug of spin down residue while sample processing. After spinning, it is better to transfer the sample to a fresh vial before processing.
10. Q: If I exceed the recommended flow rate will the mesh retainer at the end of the PhyTip column become detached?
A: No, PhyTip columns can be used at much higher than recommended flow rates and the mesh retainer remains intact.
11. Q: What is the maximum volume I can add to my PhyTip column?
A: For the 1000+ columns we recommend a maximum of 1 mL and for the 200+ columns a maximum of 200 mL although larger samples can be processed by aliquoting sample into multiple wells of a plate which are processed sequentially.
12. Q: What is the resin volume in each of the PhyTip columns?
A: The resin bed is 10, 20, 40, 80, 160 or 320 mL for 1000+ PhyTip columns and 5 or 20 mL for 200+ PhyTip columns.
13. Q: Why is flow rate important when operating with the PhyTip columns?
A: The critical step of Capture requires that the protein solution be given sufficient time to bind to the affinity resin. By using a recommended slower flow rate, maximum capture of the desired protein can occur because this increases residence time of the sample protein with the resin matrix. The default protocol utilizes a residence time of 6 and 18 minutes for 200 µL and 1000 µL samples, respectively. In addition, increased interaction of the captured protein with elution conditions due to slow flow rate, also allows maximum elution. This efficiency can also be increased by increasing the number of capture and elution cycles.
14. Q: What other factors are important in sample Capture?
A: Of course the chemistry must be suitable. For example, Protein A resin will capture Mouse IgG2a but will not capture Mouse IgG1. Also, the sample pH must be correct - in most cases, a sample of pH 7 is desirable for capture. For affinity resins, it is important to increase the residence time to compensate for low-affinity interactions. Purification of GST-tagged proteins using glutathione PhyTip columns requires using a capture flow rate of 60 µL/min.
15. Q: What is the recommended flow rate for each of the steps involved in purification?
A: In the Capture step, using a flow rate of 0.25 mL/min is recommended for Protein A, Protein G, ProPlus, Streptavidin and IMAC resins for the 200+ PhyTip columns and 0.5mL/min for the 1000+ PhyTip columns. For the Purify and Enrich steps, a flow rate of 0.5 or 0.75 mL/min is recommended for the 200+ and 1000+ columns, respectively. Check the supplied specification sheet for details. For Glutathione, it is known that the flow rate of sample over the resin will affect the capture potential; consequently the capture flow rate should be reduced below the recommended flow rates. A useful flow rate is 0.06 ml/min for Capture. (See also Q 13 above).
16. Q: Why are the flow rates for Capture, Purify and Enrich different?
A: The Capture step requires the flow rate to be slow enough to allow time for the affinity resin to bind the desired protein. However for Purification, the flow rate can be higher since the desired effect is to wash away any unbound proteins. Finally, the Enrichment step can also be performed at higher flow rates because this process involves releasing the desired protein from the affinity resin. (See also Q 13 above).
17. Q: What is the maximum loading capacity for a Protein A PhyTip column?
A: When using the largest 1000+ PhyTip column, up to 10 mgs of human IgG can bind to the Protein A affinity resin provided the sample contains enough protein and proper loading procedures have been followed.
18. Q: What are the affinities of Protein A and Protein G for any specific protein?
A: Protein A affinity resin specifically binds to most IgG’s with high affinity. In many cases, Protein G affinity resin is needed for a particular type of antibody. For example, Mouse IgG1 is captured more effectively by Protein G resin over Protein A resin. Refer to the Protein A and Protein G affinity information sheets for reference tables of antibody affinities.
19. Q: If my antibody is very dilute (e.g. 5 µg/ml) and I have 5 ml of solution, which PhyTip column do you recommend and how do I recover as much protein as possible?
A: Using the 1000+ PhyTip columns, divide the 5 mL sample into 5 equal volumes of 1 mL. With the same PhyTip column, perform the Capture step at least 2 times for each 1 ml of sample sequentially. Enrich the total protein captured in the one PhyTip column in 3X the resin bed volume of elution buffer. Alternatively, the sample could be put into at least a 6 mL vial and the 1000+ PhyTip column could be used to capture the entire sample provided enough capture cycles are used. In this case, approximately 15 cycles should be used to capture the entire sample. After sample capture, process the PhyTip column in the normal manner.
20. Q: I followed all of the instructions for isolating my protein, but I see nothing on my SDS-PAGE gel, what do I do now?
A: Check for the presence of your protein in the flowthrough using SDS-PAGE. If none is present, then the sample may not have expressed properly. If the sample has expressed, then confirm that the sample pH is correct and that the sample is compatible with the column chemistry being employed.
21. Q: My IgG protein doesn't seem to be pure; I expected to see only one band but I get two on the gel, what's wrong?
A: One would see two bands on SDS-PAGE: a light chain and heavy chain. A single band is expected if the sample has not been treated to break the sample into the 2 chains.
22. Q: I've used the Rainin manual pipette but it is tedious to keep running samples - what do you recommend?
A: The PhyTip ME 200 or ME 1000 Purification Systems are low-cost, semi automated instruments that are used to simplify the purification and enrichment of samples. The systems can purify and enrich up to 12 samples simultaneously. The MEA Purification System is a low cost instrument that is completely automated. Up to 96 samples can be purified in as little as 2 hours. All of the systems have user friendly, computer controlled software to guide the researcher through the steps of Capture, Purify and Enrich. The MEA system can also perform method optimization to determine conditions for full robotic systems.
23. Q: Why do I need the neutralization buffer?
A: The neutralization buffer keeps the protein in a physiologically stable pH environment. If low pH elution is used, the protein may be stable for a short time. Long term storage requires converting the solution to neutral pH.
24. Q: How long can refrigerated Protein A PhyTip columns be stored?
A: The columns will probably last longer, but they are guaranteed to six months.
25. Q: If I let the Protein A PhyTip column dry out while I'm using it, will it affect the protein that has been captured?
A: For optimal performance, PhyTip columns should remain moist. The column will not dry out if used under recommended operating conditions.
26. Q: Can I buy PhyTip columns with other resins?
A: Yes, PhyNexus currently supplies PhyTip columns available to capture proteins including Protein A, Protein G, ProPlus, IMAC, Streptavidin, and Glutathione. Gel filtration and ion exchange resins are also available. On a custom basis, PhyNexus offers a column-packing program for resins compatible for use in the PhyTip column. Please contact PhyNexus for details.
27. Q: What if I put excess protein through the PhyTip column, does that affect my ability to capture the protein I want?
A: No, once all capture sites are full, excess protein does not affect the existing captured proteins. This process will produce an extremely concentrated purified protein when the sample is finally recovered in the small elution volume.
28. Q: Will my protein still be active, or will it denature?
A: Many different proteins have been purified and enriched using PhyTip columns, and all of these proteins have maintained their activity after the purification process.
29. Q: These Tips look easy to make, can I just make them in my lab?
A: Looks can be deceiving, but we take that question as a compliment. The columns are designed to be disposable and effective.
30. Q: Will my proteins stick to any of the polymers of the tips?
A: The polymer (polypropylene) has been chosen for its inert nature and should not interfere with the proteins applied to the PhyTip columns. Through advanced frit design and fluid flow design, the columns have very low non specific hardware binding.
31. Q: Can I re-use my PhyTip columns?
A: We do not recommend that PhyTip columns should ever be reused since we cannot guarantee that contaminating proteins will not be eluted into the new experiment. Also the efficiency of the resin will be compromised and the recovery of enriched protein will be reduced.
32. Q: Why 96 PhyTip columns in a box?
A: PhyNexus has packed 96 PhyTip columns in a box in order to maintain industry standard formats for 96-well operation (8 x 12 format). Although not all users may be operating with 96 PhyTip columns at a time, the PhyTip columns can be removed individually and used in any format. There are packages of 48 available also as well as small packages of 12 columns designed for validation purposes.
33. Q: Will the PhyTip columns fit onto any Pipettor we have in the lab?
A: No, PhyTip columns have been designed for use only with manual LTS Rainin Pipettes, the ME 200 and ME 1000 Purification Systems, the MEA personal purification system, and a number of high throughput robotic platforms, e.g. Packard MiniTrack, Beckman FX, Tecan Temo, Caliper Sciclone, Dynamic Devices Oasis. We have chosen the tips that fit best with the most popular high throughput instrumentation in order to maximize the efficiency of the operation of protein capture, purification and enrichment.