If my antibody is very dilute (e.g. 5 μg/mL) and I have 5 mL of solution, which PhyTip columns do you recommend and how do I recover as much protein as possible?
Using the 1000+ PhyTip columns with the 20 μL bed, divide the 5 mL sample into 5 equal volumes of 1 mL. Perform the capture step for at least 2 cycles, preferably 4 cycles, sequentially for each 1 mL of sample. After column washing, elute the protein captured with 3x the resin bed volume of elution buffer. In an alternate capture procedure, the sample could be put into at least a 6 mL vial and the entire sample processed. In this case, only a portion of the sample can travel through the column in any one cycle. Approximately 15 cycles should be used to ensure that the sample protein travels through the column at least once and is captured. After sample capture, process the PhyTip column in the normal manner as described above. The first sequential procedure is preferred.
I followed all of the instructions for isolating my protein, but I see nothing on my SDSPAGE gel, what do I do now?
Check for the presence of your protein in the capture flow-through solution using SDS-PAGE. If none is present, then there are only two possibilities. Either the sample has not expressed and there is none to be captured or the sample did express and was captured but did not elute from the column. Highly expressed samples can sometimes be detected directly on the SDS-PAGE.
Assuming that sample has expressed, then confirm that the sample capture pH is correct
(approximately 7.4) and that the sample is compatible with the column chemistry being employed. Also, increase the elution strength with parallel experiments to study the effect on recovery. Many users use a known standard protein with known characteristics. The sample and standard are processed in parallel whenever a new unknown protein is being expressed and/or purified for the first time.
I am using the PhyTip ProA tips to isolate Mouse IgG, but I don’t see any bands in my gel.
How do I know that my system is working?
Review the expression system and the type of mouse IgG in the sample. Refer to Protein A and
Protein G affinity information sheets for reference tables of antibody selectivities for affinity resins. Check the flow-through from your capture steps to verify that the protein was captured.
Check the wash conditions to make sure the specific protein of interest was not removed in the wash step. Next, check to see if the protein is still attached to the resin after the final elution step (pH of the buffer may not have been low enough). Finally, check to see if the eluted protein neutralized effectively and did not denature. Many users run an antibody standard in parallel with the sample to help monitor loading and recovery performance of the sample.
What if I put excess protein through the PhyTip column. Does that affect my ability to capture the protein I want?
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.
What is the maximum mass that I can get from a 20 μL ProA, ProG and IMAC column?
What about other bed volume columns?
The PhyTip process can often capture more protein onto the resin bed than suggested by the original resin manufacturer. The repeat cycling of the sample capture process used by the
PhyTip column maximizes resin performance. Multiply the resin capacity per unit volume by the number of μL in the column bed.
Can I recover more material if I do two elutions rather than one?
Experiments suggest that with a single elution step (and depending upon the affinity of the resin and the elution strength of the elution buffer) anywhere between 60 and 80% of the purified protein can be removed in a single elution step. This percentage can be increased with a second or third elution step if the end result is to recover more mass. But this will be at the expense of concentration if the elution volumes are combined.
How do I know that all of the protein has been eluted from the resin?
Elution of protein from the tip can produce two different end results. If the requirement is for highest concentration then the minimum volume of elution is recommended. However, this may result in a lower percentage of purified protein being removed from the column. If the goal is maximum mass of purified protein, then several wash steps will ensure maximal removal of protein from the resin. This however will reduce the concentration of final protein. It is unlikely that 100% of protein will be removed from the resin, and researchers can expect that with sufficient elution, approximately 95% of protein can be removed from the resin.
I have heard that you can elute from an IMAC resin using variable pH. Is this true?
Yes, low pH elution buffer can remove a His-tagged protein from an IMAC resin. In addition, adding a chelator such as disodium EDTA to the elution will remove the protein from an IMAC column but will remove the nickel metal as well.
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?
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.
I am running an IMAC resin and my final product is not as pure as I had hoped. How can this be improved?
Wash buffers for IMAC resin will often contain low levels of Imidazole that help wash away some of the nonspecific 2, 3, 4 or 5 His tagged variants that may have been expressed along with the specified 6-His product. Imidazole in the wash helps remove any histidine rich protein in your sample that competes for Ni(II) in the resin. Sometimes the concentration of the imidazole is not high enough to remove these contaminants (the wash is not stringent enough) so the final product will not appear as pure as it could be. But care must be taken if the wash buffer strength or stringency is increased because the sample protein of interest might also be removed in the wash. If the wash stringency is too high, the protein might be very pure, but the yield might be very low.
Why do you recommend two different types of wash buffers while using ProA or ProG resin. What is the second buffer and what does it do?
Two wash buffers enhance the elution step by reducing the pH shock when moving from a pH 7 wash buffer to a pH 2 elution buffer. Low pH elution buffer has higher strength to break the binding between resin and protein and can improve protein recovery. If there is no intermediate saline wash, the residual pH 7 wash buffer that resides in the resin will decrease the pH strength of the elution buffer which then results in a lower yield of eluted protein. Adding an extra step to the process removes the buffering effect of the first wash step and enables the low pH of the final elution step to be effective and produce the highest recovery of purified protein. The second wash can be very important to achieving reproducible elution of the protein and is always recommended.
What is the minimum recommended wash buffer volume that still achieves the highest purity of sample?
This will depend upon the volume of the resin in the tip, the type of protein being purified, and the types of contaminants that are mixed in with the protein of interest. For example, a 200 μL wash of a 5 μL resin bed will probably produce the optimal purity results. It is possible that the same level of purity could be achieved using a 100 or even a 50 μL wash volume, but given the cost of the wash buffer and the difference in time between a 50 and a 200 μL wash, the recommendation is to go for the highest volume rather than try to compromise.
Is imidazole in the equilibration buffer and sample buffer? If so, why?
We recommend using the following equilibration buffer while using IMAC resin: 50 mM sodium phosphate, 1.5 M sodium chloride, 25 mM imidazole, pH 7.4 Note: This is a 5X buffer and needs to be diluted to 1X with water before use. Imidazole is present to clean the column from any material that might bind to Ni present on the column. Imidazole in the sample buffer can help keep the protein pure by limiting the capture of impurities along with the recombinant sample protein.
What is the difference between high and low stringency buffers?
Low stringency washes remove weakly bound nonspecific impurities. If the sample protein is large and bulky, then the column should be washed with low stringency buffers to avoid losing sample protein. Higher stringency washes can be used for tightly held sample proteins. For IMAC resins, the difference between the low and high stringency buffers is the concentration of imidazole used. For IMAC, a low stringency buffer is 5 mM imidazole and a high stringency is 20 mM imidazole. For ProA/ProG resins, the stringency of the wash can be controlled by the volume of the wash. Smaller wash volumes provide a low stringency wash. Large wash volumes provide a higher stringency wash. Stringency for these resins can also be controlled by the wash pH (lower pH is a higher stringency wash), but this should be done carefully with testing to avoid sample protein loss.
What is the relationship of high protein recovery and high protein activity?
Interestingly, high protein recovery and high protein activity are not necessarily correlated showing the need for method development. Using the PhyTip columns operated in parallel, it is quite easy to study two or more variables, such as the effect of pH and salt concentration, in the same set of experiments. The complex interactions of sample and contaminants with the resin can be studied to optimum yield.
In protein purification, not only is yield important but the recovered protein must remain active and not agglomerated. In one experiment of parallel method development for purification of an antibody on one type of Protein A column, different conditions of capture, wash and elute (8 each) were tested for each of 4 clones. The recovered proteins were screened for yield (ELISA) and retention of activity (SPR). The results show that high recovery and high (retained) activity are not correlated and both goals should be considered when developing separation conditions for proteins. It is difficult to consider these goals without parallel purification.
When separation conditions are established, parallel operation of columns can be used to separate up to 96 samples at-a-time.
Why do I need the neutralization buffer?
If low pH elution is used, the protein may denature if left at low for an extended period of time.
Although there is no set time period, the protein buffer pH should be raised to neutral pH in an expedient manner. The neutralization buffer is concentrated and can be added directly to the eluted protein to raise the pH to a physiologically stable environment. When necessary, confirm the pH has been raised to 7 after an aliquot of the concentrated neutralization buffer has been added to the eluted sample.
Will my purified protein still be active or will it denature?
PhyTip tips are designed to have ultra-low non polar surface area that could come in contact with proteins. Proteins may unfold or denature when they contact a non polar surface. This is especially true for membrane proteins because these proteins have a large non polar component to their structure. Limiting the amount of non polar surface in the PhyTip column helps keep proteins from denaturing. PhyTip purified proteins are likely to maintain their activity during and after the purification process.
What is the optimal elution pH for a Protein A or Protein G tip? How quickly should I neutralize the eluted protein? How should the eluted protein be neutralized?
The optimal pH will vary depending upon the protein that’s captured and the affinity it has for the resin. Smaller proteins stick tighter to the resin. Obviously, the gentlest conditions are desired for elution. Most proteins will elute from the resin at pH 2.5 – 3.5 but it may be necessary to elute as low as pH 2.0. See customer support for the types of low pH buffers used for elution. After elution, neutralization with a buffer is recommended to bring the pH back up to neutral as soon as possible – no longer than a few minutes. The neutralization buffer may be a pH 8 or 9 depending upon the strength of the buffer used to elute the protein.