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December 29, 2017

Questions and answers from ÄKTA club web chat

By Lotta Hedkvist, Product Manager ÄKTA systems at GE Healthcare

Your protein purification questions discussed with GE Healthcare R&D scientists


In our web chat on October 18, ÄKTA system users asked questions relating to lab-scale protein purification and ÄKTA systems, which our GE experts answered during the live session.

The expert panel included scientists from our R&D team in Uppsala and from Scientific Support. Questions about chromatography techniques, troubleshooting tips and tricks, and the use of ÄKTA systems were submitted in writing by protein purifiers globally.

We have included the questions that were answered, plus a few additional ones that we did not have time to answer during the web chat.

We look forward to the continued discussion of your questions live during our coming web chats.

Do you have additional questions?
Feel free to continue the discussion about lab-scale protein purification and ÄKTA systems on our Discussion Forum at proteins.gelifesciences.com/forum

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ÄKTA club is an online platform for ÄKTA system users enabling easy and quick access to trustable information about ÄKTA systems and protein purification. Connect to forums on ÄKTA club. Share your experience and results, engage in inspiring discussions, and learn from peers and GE experts.

We are curious to know if it's possible to connect an external pump to ÄKTA pure. We are attempting to simulate sample flow rates, which are higher than the limits of the sample pump. Furthermore, could one program to fractionate while stile accepting flow from an external pump? My colleague and I wish to use a faster flow rate than allowed by the system and sample pump. Can we connect an external pump and still have the system perform fractionation somehow? We would likely need to make our own phases, allowing the system to fractionate based on time, correct?

You can use ÄKTA pure together with the I/O box E9. To start the external pump you simply send a digital signal (TTL, open/close circuit). There are also two channels for an analog output with a range of +/- 1000 mV. To say if this is suitable for controlling the external pump is difficult without knowing the specifications. In practice yes, you can run at a faster flow rate but this is not recommended since the system has been tested up to 200 bar (20 MPa, 2901 psi) at the specified flow rates. We cannot guarantee what will happen using the higher flow rates—this also goes for the fraction collector. In short, this has not been tested. Sending a start signal for the pump via the I/O box E9 is possible.

Where is the best place to zero or blank the A280?

I recommend that you auto-zero the UV signal at the end of the equilibration, i.e. just before the sample injection.

Hi. This is very simple question. When we want to use ÄKTA systems, which part do we need to switch on first—the chromatography system or the computer?

Hi, I usually switch on the ÄKTA system first and then the computer but you can do the opposite as well.

How to make sure our ÄKTA systems are well maintain even after 10 yr of service? For example the pcb slave board starts to malfunction etc.

I recommend that you perform regular maintenance and do not leave the system for long with buffer that can cause bacterial growth or salt precipitation. Contact you GE service representative (gelifesciences.com/contact) to get support for this.

We recently purchased ÄKTA pure 25 instrument. I am here to find out what resources we currently have for training and improving our knowledge of the system.

I suggest that you contact the GE representative who will perform the training and discuss what is planned or if you have any special requirements. There may be differences between different countries.

Even though I blank the A280 at equilibration before sample injection, often the zero is quite high - how can I prevent this or make the zero more real?

Hi, are you running a gradient and is there anything in the mobile phase that absorbs UV light? In such case you could try to zero the UV when you have a mixture of A and B buffer in the detector cell.

As I understand a remote license allows me to use Method editor, Evaluation module as well as system control. If I buy such a license, can it be shared by two people on the same network - one using it to control a system and another using it to write methods or analyze data?

A remote license is a floating license that can be used by any user on the network but only one at a time.

Me and my colleagues are working on similar projects and want to share methods but I also want to make sure no one messes up my work. What is your advice?

Oh, that is an interesting question. In a network environment, you have excellent possibilities to share your work. You can define who should have access to a specific folder. You can sign and lock methods and results and you can set general access rights for those who can edit global objects such as phases in phase library. My advice is to store methods that you all need access to in a common folder. If you want others to have access to a method but without the possibility to edit, you can sign and lock the method. Other users can then open the method but need to save their own copy if they want to edit it.

How can I write a design of experiments (DoE) method in UNICORN?

Thanks for the question. A DoE design is easily created for a method in the Method editor of UNICORN. Simply click on the "design cube" icon or select Design of experiments under the Tools menu. This will take you to a dialog where you define factors, responses, and select the appropriate design for your objective.

How can I access to my statistical analysis of DoE run in ÄKTA?

Running the experimental method that includes a DoE design will create a "DoE result" with the specific cube visible in the result name. After adding response values to the data table, the modeling is performed directly when clicking the analysis tab. The statistical evaluation from the modeling is available in the analysis tab where the model can be edited and the statistical evaluation can be viewed in the different reports. Response values are added one by one or by copy paste from for example Excel®. In UNICORN, I would add the response values to individual copies of the DoE result file in order to get two separate models for each response.

We've heard that you can use "watch command" or something like that to get an ÄKTA avant to collect fractions during a load/wash. This is for a run where our protein of interest passes through, while contaminants bind. Can you explain this please?

There is a function in UNICORN called Peak fractionation that you can use to start and stop fractionation depending on the level or slope of the detector signal. In the instruction, you specify the fractionation volume. This instruction will make fractionation start when a peak is detected and stop when the end of the peak is detected. You also have the possibility to use a watch command on the detector signal. You then specify what triggers the watch, i.e. a slope or level that is higher or lower than a specified value. An action is executed when the conditions set to trigger the watch are reached.

Can ÄKTAexplorer be upgraded to UNICORN 7? If an upgrade is possible do we need to upgrade the computer as well?

Yes, ÄKTAexplorer is supported by UNICORN 7. You may need to upgrade you computer if it is old/slow. You can find the requirements on the computer on our web page, gelifesciences.com/UNICORNPCspecifications

I usually borrow my colleagues' ÄKTA pure when I need to purify my biomolecules before the next step in my research. I want to quickly know how much I have in my fractions. What I usually do is to go to a spectrophotometer to measure the UV absorbance. Can UNICORN help me with this in a more straightforward way?

I am happy to tell you that UNICORN can calculate the amount and concentration in your fractions if you know the extinction coefficient of your biomolecule. UNICORN keeps track of your fractions and if you plan to pool several fractions to get more material, UNICORN can help you calculate the amount and concentration to assist in your decision of which fractions to pool. In UNICORN 7 evaluation software, we have included short videoclips showing how to use the software. I recommend you to have a look at these.

Can ÄKTAexplorer do buffer scouting?

In ÄKTAexplorer there is a function for buffer preparation (BufferPrep). This function is however not supported in UNICORN 7.
I would very much recommend that you use ÄKTA avant and the BufferPro function for buffer scouting since that function has automatic compensation for ionic strength and you can set the desired buffer concentration in the method. This function also allows you to mix buffers using corresponding acid and base for common buffer substances. If you use a wide range buffer, you can scout on different pH and buffer concentrations automatically, for example, to find the best conditions for an ion exchange run.

What is tandem purification?

Good point! Not that obvious from the name but we mean running an automated two-step purification using two columns connected in series. Elute from the first column directly on to the second column.

What do you mean by extinction coefficient of your biomolecule? I was wondering if I can use UV absorbance at 260 or 280 nm and calculate the concentration? Is that possible? Is there any relationship between these absorbance values? I usually use UV280 to see if the protein is binding to my column or eluting but I don't know what information I can get from other UV absorbance values available in UNICORN?

Yes, in UNICORN 7 you can use the UV absorbance to calculate the concentration since the concentration is proportional to the UV absorbance according to Beer´s law. What you need to know is the UV cell length and the extinction coefficient for your protein at the given wavelength. If you are using an ÄKTA avant or ÄKTA pure system, UNICORN will know the cell length and for other systems you can enter cell length manually.
Beer´s law:
Absorbance=e*l*c
Where e is the extinction coefficient, l is the UV cell length, and c is the concentration

What is the best way to manipulate proteins between nickel purification and a cation purification exchange for example?

If your purification steps include an IMAC run with Ni Sepharose followed by a cation exchange step, you will need to change the buffer of the protein that was eluted in IMAC. Our recommended elution buffer for Ni Sepharose is 0.5 M NaCl, 0.5 M imidazole, pH 7.4. However, this pH and conductivity are not suitable for a cation exchanger. Therefore, you will need to exchange (using HiTrap Desalting column) to a buffer with no imidazole, low salt, and appropriate pH of at least one pH unit below the pI of the protein before loading to a cation exchanger.

After a purification run of E. coli lysate on my HiTrap column, there is yellow colorization and backpressure increases. What should I do to restore the column?
When cleaning with 1 M NaOH, is it better to first strip the column, NaOH treat, and then recharge?

A yellow colorization of resin after applying a complex cell extract may come from many different contaminants. Cleaning with 1 M NaOH with the reversed flow direction is the best method if the contaminants are hydrophobic. For very hydrophobic proteins you may also try using 70% ethanol or 30% isopropanol with reversed flow direction. Cleaning with 1 M NaOH works for many different types of column, not just IMAC columns. For IMAC columns such as HisTrap HP, one can try to clean with 1 M NaOH without recharging if you use same type of sample. Otherwise you can strip the metal ions with EDTA followed by cleaning with 1 M NaOH. Finally recharge the column with the metal ion of your choice. For other IMAC columns such as HisTrap excel, it is very difficult to strip metal ions because they bind very strongly to the ligand of the resin.

How can I best get clean tag purification off the HisTrap column when increasing wash stringency? I only see increased loss of the protein of interest.

If I understand your question, you are losing your target protein when increasing the imidazole concentration. That is normally the case and it is a trade off between purity and yield. If you increase the imidazole concentration, the purity will increase but the yield will decrease. Therefore you need to decide which purity and yield you find okay. If you need more yield and the purity is not okay and you need to add a second purification step with either SEC or IEX column. You can also try to use HiTrap Talon® or HisTrap excel where you will get higher purity. However the capacity of these columns is lower than with HisTrap HP.

I am using GE columns for purification of His-tagged protein in 5 mL HisTrap HP or HisTrap FF column scale. Using the same conditions, HisTrap HP column worked fine but FF column did not. With FF, there is lower binding as well as binding of host protein. I would like to scale up from 5 mL to 20 mL column or more. We tested 20 mL FF column as well but did not get satisfactory purification, connecting 2 × 5 mL HP columns worked better. Can we connect 4 HP columns, or is it too much? Does the HisTrap HP column gives better purity that the FF?

Connecting four HisTrap HP columns should be possible according to our packing expert. Do not exceed maximum operating back pressure (this may mean that maximum flow rate cannot be used). We have seen that HisTrap HP in many cases has higher capacity in comparison with HisTrap FF, which is confirmed by your results. If you still want to test HisTrap FF further, the advice is to increase imidazole concentration during equilibration and sample application and wash to get rid of the host proteins. HisTrap HP will produce better resolution than HisTrap FF. Smaller beads give more concentrated peaks. Always use HP when sample is already clarified or in a second step of purification.

What are the best steps to clean up a nickel purification using the HisTrap FF. Is there a better column?

HisTrap FF may be cleaned with NaOH but only after stripping Ni2+ ions. Recommended stripping buffer: 20 mM sodium phosphate, 0.5 M NaCl, 50 mM EDTA, pH 7.4. Strip the column by washing with 5 to 10 column volumes (CV) of stripping buffer. Follow this by washing with 5 to 10 CV of distilled water. Remove precipitated proteins, hydrophobically bound proteins, and lipoproteins by washing the column with 1 M NaOH, contact time usually 1 to 2 h (12 h or more for endotoxin removal). Then wash the column with approx. 10 CV of binding buffer, followed by 5 to 10 CV of distilled water. Isopropanol 30% or ethanol 70% can remove very hydrophobically bound proteins.

In what cases would HisTrap FF be better than HisTrap HP?

Very good question! HisTrap FF should always be used in a first step of purification, for capturing your His-tagged protein and having the maximum of binding capacity. HisTrap HP will be used when sample is not crude such as after a first step of clarification or purification.

How many recharges can we do before column function begins to degrade? Is changing back pressure the main determinant for getting a new column? At what back pressure would be a good time to retire a column?

HisTrap column life time is really a function of the quality of your sample and how you filter it. Whatman syringe filters are highly recommended to avoid clogging. Most of the time, the column resin can be reloaded many times before back pressure starts to increase. Back pressure is one thing you can check to find out when it is time to replace a column, but also the performance of the column in general. If the output, for example yield of target protein and purity, is decreasing and cleaning the resin does not help then it is time to change column. However, you should always try to clean the column first to see if you can restore the column. HisTrap column has a limit pressure of 0.5 MPa. Depending of the system configuration (back pressure generated by the system itself) or buffer composition and temperature, this maximum pressure could be reached or not. Sometimes, the flow direction in the column may have an effect on the pressure and reversing the flow might help to decrease the general back pressure somewhat.

I get good purification from eluting tagged proteins from HisTrap FF. However, the protein crashes in the column when I attempt an on-column tag cleavage. How can I prevent this when adding detergents?

Try 5% to 10% glycerol. If that does not work, you may need to decrease the salt concentration or load less protein to the column with a lower concentration of protein bound to the column as the output.

I am using GE’s HisTrap HP to purify my His-tagged protein. Binding buffer: 20 mM sodium acetate, 200 mM NaCl, 5mM imidazole at pH 5.2. I elute protein with the same buffer plus 500mM Imidazole, but my protein is mostly in the flowthrough. What can be the problem?

His-tagged proteins bind to the resin in HisTrap HP through the histidine residues in the tag to bind to the Ni2+ ion on the resin, and for that purpose the imidazole side chain in the His residue needs to be deprotonated. The pH of your buffer (pH 5.2) is too low, the purification should be carried out at a slightly basic pH (say 7.4 to 8.0). The recommended pH in the instruction for HisTrap HP is pH 7.4.

I have a problem: When I purify simple chain fraction variable IgG (∆CH1) my protein always precipitates. I elute fractions with 0.3 mL of 0.1 M glycine buffer, pH 3 and I collect the antibody with 0.3 mL of 0.25 M Tris HCl to neutralize and diminish the concentration. Furthermore, I decreased the load of antibody on the protein G column, but the precipitation continues. I observed that this protein with concentration more than 0.3 mg/mL precipitates. What can I do to improve the purification?

Hi, when does the precipitation occur? On the column or when you are adding the Tris-HCl? I assume that you are neutralizing the fractions with your IgG immediately after elution to avoid denaturation of your IgG. You need to optimize the buffers you are using. Add 5% to 10% glycerol to avoid precipitation, change the buffer you are using, and decrease salt concentration as that may be the reasons for precipitation.

I can´t purify dog IgE antibody types with kappa chain using HiTrap Protein L, my antibody is purified in the flowthrough. How can I purify this antibody?

Hi, very interesting question! From other user's feedback, antibodies from dog do not seem to bind very strongly to protein L. We have another resin named KappaSelect, which could be tested but we have no data for binding capacity for antibodies from dog.

Are the MabSelect PrismA column flow rates on par with previous MabSelect columns? What other differentiators are there and how do you hope to address any shortcomings with the new columns.

Hi, you can find all information about MabSelect PrismA that is available on the web page: http://gelifesciences.com/mabselectprisma

I am using nProtein A Sepharose FF for immunoprecipitation experiments. Is it possible to use SDS with the sample?

Yes, in certain very stringent protocols (like that used in chromatin immunoprecipitation) up to 0.1% SDS is often used. However, this may affect the antibody-antigen interaction and so it must be tested out first. SDS may also disrupt the protein conformations and denature proteins so use with care.

How can I optimize the running conditions to obtain the best resolution possible with size exclusion chromatography?

After selecting a resin that has the best fractionation range for your protein, you should lower the flow rate and sample volume to improve resolution. The buffer conditions affect the chromatography result since the environment for the protein is important. Avoid any interaction with the resin or other compounds in the sample. It is also important to configure the system you are using. For maximum resolution, the system volumes should be kept at a minimum. Short, and narrow capillaries should be used and unnecessary system components should be bypassed.

Sephacryl S-200 HR column is being used—the issue is that the smallest proteins are expected to elute out before 1 column volume (CV), usually by 0.7 CV. There is a delay in the elution of the protein. For example, lysozyme, a 14 kD (Mr 14 000) protein usually elutes before 1 CV in the first few runs, but then it elutes out after 1 CV. What might be the reason? Please suggest how I should go about troubleshooting.

Thank you for the question. What you see is nonspecific binding to the column, which delays the retention of the molecules of interest. If you use the same mix of the protein, an external, unknown factor might have caused the protein to elute late. The best way is to store several aliquots of your sample mix in the freezer which you can take out before each run. Then you will have fresh sample and also the same mixture of sample. Do not use the same sample mix for days as there might be changes. Check the column pressure during the run as well. If it is high, you need to clean the column. It is also recommended to clean the column after 10 to 20 runs depending on how dirty the sample is. In general, to minimize nonspecific binding, it is a good idea to test the buffer conditions by changing salt concentrations or adding organic solvent such as glycerol or detergent.

Do you guys have experience with IgG interacting with columns such as Superdex 200? What would you do differently to purify the protein?

Hi, I assume you mean that different IgGs are retained at different times on the SEC column. The reason is that IgG can have different characteristics in form of hydrophobic, aromatic, and ionic groups on the surface. Such interactions may lead to an analyte eluting later than expected and could give the appearance of a lower molecular weight. To overcome undesirable secondary interactions, it may be necessary to perform method optimization, which means that you may need to change the salt concentration or add organic modifiers such as glycerol or detergents in the elution buffer. Adjustment of the ionic strength, pH, or organic modifiers will change the conditions in the SEC runs.

How large volume of sample can I inject to the SEC column without losing resolution?

We recommend injection of no more than 0.3% of the total column volume if you need highest resolution separation between two proteins that are close to each other in size.

I am working with liposome-encapsulated protein (Mr 60 000). I am trying to separate unencapsulated protein using GE chromatography columns. How can I optimize the method? Which column do you suggest? Does using the column affect final protein concentration, in other words affect the dosage?

Hi, optimization of your method is recommended but it depends on your objective of course. Sepharose CL2B (SEC) column is a good starting point. In most purification steps you will lose some material but this all depends on what you are purifying and how. Nonspecific binding can sometimes be a hassle that will have effects on recovery. A quick search using a web browser gives for example this article.

How often do you recommend cleaning a size exclusion chromatography column?

It depends on the status of you sample but in general we recommend you clean the column with 0.5 M NaOH after 10 to 20 separation cycles. If you have dirty samples, perform the cleaning after even fewer cycles. Always clean the column if you are going to change type of sample to avoid contaminations of foreign compounds.

When using Superdex 200 10/300 GL on my ÄKTAFPLC, I occasionally get a strange baseline immediately after injecting. It starts high, over 600 mAU, and very slowly decreases over about 10 mL. I have cleaned the column, degassed all buffers, filtered my protein, and equilibrated with multiple column volumes but still this baseline appears. I know its not protein because I have run it on an SDS gel. Have you experienced this issue before?

Hi, thank you for the question. Run the system by bypassing the column and turning the injection valve to sample injection to check if the system used is causing the problem. One reason might be that when you start the injection, a small pressure increase occurs, releasing precipitated contaminants into the flow path that are detected by the UV detector. If that is not the case, precipitated contaminants from the column might have been released and detected during injection. It is always important to clean all parts in the system before or after use.

Which columns would you suggest for purification of peptides?

Hi, If you want to separate by size I recommend that you use Superdex 30 Increase columns which replace the Superdex Peptide columns. Otherwise, RPC columns are normally the most commonly used column type.

In this list of HiScreen columns that someone "must have", do you see redundant items? HiScreen Capto adhere 4.7 mL 28926981, HiScreen Capto adhere ImpRes, 1 × 4.7 mL 17371520, HiScreen Capto MMC ImpRes 1 × 4.7 mL 17371620, HiScreen Phenyl HP 1 × 4.7 mL 28950516, HiScreen Butyl HP 1 × 4.7 mL 28978242, HiScreen Capto MMC 4.7 mL 28926980, HiScreen Butyl FF 4.7 mL 28926984, HiScreen Phenyl FF (High Sub) 1 × 4.7 mL 28926988, HiScreen Butyl-S FF 1 × 4.7 mL 28926985, HiScreen Octyl FF 1 × 4.7 mL 28926986, HiScreen Phenyl FF (low sub) 1 × 4.7 mL 28926989?

This list contains HiScreen columns with HIC and multimodal ion exchanger resins. It includes multimodal anion/cation exchangers and HIC resins with different hydrophobicity, on agarose resins of different particle size. Each column has some properties that differ from the rest, so there is no "redundant" item on the list.
There are also different selection kits that contain a selection of HiTrap columns, prepacked with different chromatography resins for screening to get optimal performance and conditions to use for application and development work. Several products apply, for example:
HiTrap IEX Selection Kit, product code 17600233
HiTrap Capto IEX Selection Kit, 28934388
HiTrap HIC Selection Kit, 28411007
There are also other products for screening purposes, column formats, and multiwell plate formats. Please check with your local sales representative (gelifesciences.com/contact) for more guidance to fit your application purposes. I would go for the screening kits prior to buying the HiScreen columns. Good luck!

I am using HiTrap Capto Q for protein purification. Should I add a HIC column before Q or after Q for removing impurity?

Without knowing more detailed information on targets the answer becomesa bit of a guess. But generally, the capture is beneficial using a technique that will allow for a wash step to clear out most of the impurities. Using a HIC after IEX will be beneficial since the latter starts with a low ionic strength and a pH depending on protein and IEX type and ends with a high ionic strength and/or pH changed. HIC starts with a high ionic strength (addition of salt required) and ends at a low ionic strength. HIC is more frequently used in intermediate and polishing purification steps.

Should I optimize my sample application part (capturing target protein ) on a Capto Q column first and then optimize the elution part? Or should I optimize both capturing and elution at the same time. I found that the number of parameters will be increased if I have to optimize both steps together.

A more frequently used approach is to screen critical parameters in high throughput experimental formats (96-well prepacked filter plates or PreDictor RoboColumn units). Doing this allows you to screen parameters for both the capture (loading) part and the elution part. Once the critical parameters are known and a rough estimation of parameter ranges is set, you can follow up with an optimization study on the column.

I run DoE, it contains 19 runs. I noticed that the column changed to a pink color and never changed back to its original color even after cleaning and stripping steps between runs. I used 50 mM MES + 50 mM ammonium acetate + 1 M NaCl with pH of 4.0 for the stripping step. I thought that this is a strong buffer in terms of conductivity to remove all the impurities, but I am not sure why the color of my column changed and did not go back to normal? I also want to know if I can trust the results and do the analysis or if there might some carryover from previous runs?

An instruction/protocol is supplied with all our columns and this provides information about their recommended use. A recommended cleaning procedure is also included in the instructions/protocol. For Capto Q, this includes the use of both 2 M NaCl and 1 M NaOH; please check the protocol to see details. Carryover is sometimes a problem with "sticky" samples, and in this case, a thorough CIP of the column between runs is always recommended.

I have two proteins with similar pI's and charges at a given pH. One of them purifies on HiScreen Capto SP ImPres very well. The other seems to disappear and recovery is drastically less than input. What can be the cause of this, and what is a possible solution?

Always interesting with these type of application inputs. Its difficult to answer directly without more information. There is always a risk that the protein does not release from the column at the used elution conditions. Performing a CIP of the column and collecting what comes off could reveal if material is still bound after elution. There is a possibility that you are not getting the protein out from there. Screening studies of several chromatography resins could reveal which resin is most appropriate for your application. You can select different experimental conditions in your screening as well as CIP.

Hi, I am using HiTrap Capto Q column to trap an antigen from a fermentation broth. There are many factors that I need to optimize but I would like to know should I optimize binding of target protein on the column first and then start to optimize the recovery or elution of the target protein? Or should I perform both optimization steps together?

Hi, thank you for the question. I recommend that you optimize the binding conditions first as there are often practical reasons for dividing the experimentation into a separate sample loading and a separate elution study. You may have critical parameters such as contamination, pH etc. that may be important during binding but are not as critical for the elution. It is also important to optimize the wash step. A more frequently used approach is to screen critical parameters for both the capture (loading) part and the elution part in high-throughput experimental formats (96-well filter plates or Predictor RoboColumn units) and design of experiments. You can use small-scale columns as well. Its just that you can be more time efficient when using a high-throughput format. Once the critical parameters are known and a rough estimation on parameter ranges is set, you can follow up with an optimization study on column. Identifying the most critical parameters can reveal that they are present both during sample loading and elution.

Should I add a HIC column before a Capto Q IEX column or after it to increase recovery and also perform a scale-up study and later use a bigger column? Which approach is more applicable based on your experience?

Good questions! The use of IEX before HIC simplifies the sample adjustment before HIC, since salt is used for elution in IEX and because it is acceptable in HIC (and since additional salt is added). However, if ammonium sulfate precipitation is performed before chromatography, it is usually convenient to apply HIC as first purification step (before IEX) since HIC requires salt for binding anyway. Buffer adjustments may be required. After a HIC step, the ionic strength will usually have to be reduced in the sample before application to the IEX column. This can be done by desalting using size exclusion chromatography (HiTrap Desalting column or similar) or by dilution. Dilution will mean handling of larger volumes and other additional steps needed (buffer exchange, buffer conditioning, etc.) will affect sample handling times and the overall purification efficiency.

How can I best optimize parameters to get the cleanest, most concentrated elution off either the HiScreen Capto SP ImpRes or the Capto Q ImpRes?

The first thing you need to do is to screen the load, wash, and elution conditions you need. If you know the pI of you target protein, set a pH where your protein binds to the resin. Then you should run an elution gradient to find out which conditions (salt conc.) the target protein is eluted at. When you know that, you can then optimize by using stepwise elution with a salt concentration during wash that is below the concentration where your target protein is eluted. This approach will remove contaminants. Afterwards, increase to a salt concentration that elutes the target protein but not other contaminants that need higher salt concentration to be eluted. You can also set up a design of experiment where you define factors such as salt, pH, ionic strength, and buffer. You need also to set the responses such as yield and purity.

Can I purify membrane proteins by ion exchange chromatography (IEX)?

Hi, yes of course. However it is important that you are using detergents in the buffers and sample. You need to optimize the running conditions before the purification as well.

I am working on optimization of a method using HiTrap Capto Q, 1 mL column. In order to scale up my method, should I increase my column size to 5 mL and then 20 cm packed column or can I jump from a 1 mL column to a 20 cm column for scale up?

I do not know the real objective, if the purpose is to produce material for research or if it is for scale-up studies (going towards production)? But one important thing to keep in mind in scaling up is to maintain the used residence time on the column. Experimental studies using flow rate as a factor can reveal if the binding/elution is flow-rate dependent.

Do you have equivalent solutions (columns) to perform the separation by HIC and double RPC steps reported in this paper (contortrostatin isolation): "(Poly LC, Columbia, MD) was used for hydrophobic interaction HPLC. Cl8 (218TP54 and 218TP510) columns were used for reverse phase (RP) HPLC (Vydac, Hesperia, CA)" from Thrombosis Research 73, 39–52, 1994.

Hi, Based on the method in the publication we do not have RPC in our portfolio. We have different HIC products but you need to screen them to see the one that will give similar output as polypropyl aspartamide.

I've heard about 3D printing in chromatography. Can you tell me more about this? I'm curious to know if it's something I can use.

Additive techniques are emerging in different fields. GE is using additive manufacturing as a key part of the evolution into a digital industrial company. We will leverage the use of additive techniques for design and creation of lighter, stronger parts and systems and products that improve performance that are more complex in design, yet simpler to build.

What do you advise for cleaning columns?

Hi, good general question! it is very important to be able to clean the column to keep it in good condition and to extend its life. In many cases, NaOH could be used but when a ligand is attached to the beads, this ligand could be more labile. Cleaning steps are always described in the instructions supplied with our columns or in online instrcutions available from our website.

I have a problem with during polishing using SP Sepharose resin using a 100% linear gradient. How do I create a step gradient?

Without knowing more about the specific application it is difficult to answer. I mean several problems could apply, please respond to your local representative (gelifesciences.com/contact) to file a Tech support case so that we can dig into the problem in more in detail.

What are your tips to increase the purity of enzymes?

Purity is always one of the most important responses for our purification efforts. Increasing purity is for me a matter of optimizing your method. A strategic approach is a good choice; design of experiments (DoE) included in UNICORN software is good for setting up and evaluating optimization studies.

How can I test column integrity for a preparatory-scale column?

The column integrity can be check by measuring asymmetry factor and the theoretical number of plates/m. These two parameters are related to the packing quality of the resin. But unfortunately, system configuration can result in numbers different of those written in our instructions. This is not an issue if you always use the same system. But the general rule is to always try to minimize system volumes by using short tubing before and after the column and bypass all unnecessary valves.

How many times can you reuse columns?

It depends on whether you are using the same sample on the column and how crude the sample is. For HiTrap columns, normally we say that you can reuse one column 5 to 8 times when loading the same sample, then the column should be cleaned. When using different kinds of samples, you may need to clean the column after each use or use a new one.

What's the lifetime use for HisTrap and HiScreen Capto columns?

It depends on the product. An expiry date is written on the side of all HiTrap and HiScreen columns. You can also find information on our web page. However, it is also dependent on how you have used the column and the environment. If dirty samples have been used and the column has not been cleaned according to our recommendations, the column lifetime is shorter. Columns should be stored cold. Storage in room temperature reduces the column life. You should always follow our recommendations in the instructions and you should always store the columns in 20% ethanol if nothing else is stated in the instructions.

To purify RNAP I run a HisTrap HP with multiple step washes, anion exchange, and then Superdex 200 but in the final SDS gel there are many faint residual contamination bands. I have tried to optimize the HisTrap HP column by using higher concentration imidazole washes, doing a shallow gradient elution on anion exchange with 0.5 mL fractions, and running a smaller load volume for the Superdex but these bands still persist. Since my protein is so big I would expect the final product to be easily separated from these residual contaminants but that is not the case. Why would these not be removed after using all these columns?

Good question. Sometimes, during sample preparation steps (lysis or other mechanical treatments) proteins can stick to complex and copurify even if it is not a specific binding. And sometimes unfortunately, nonspecific binding could be very strong! Detergents, salts, or cahotropic agent (such as urea) could help to remove the impurities.

AC – Affinity chromatography