SAXS has emerged as a standard biophysical tool deployed routinely for characterizing macromolecules of biomedical interest. The relative logistical simplicity embodied in a technique that provides easy access to the size and low-resolution shape of macromolecules makes it an essential part of the biophysicists’ tool kit. With the range of separation coupled SAXS experiments, such as SEC-SAXS, at BioCAT, SAXS data can be collected from even the most challenging systems.
There are four equilibrium data acquisition strategies available at BioCAT: SEC-SAXS, SEC-MALS-SAXS, IEC-SAXS, AF4-MALS-SAXS and batch mode SAXS.
Below we give some general guidelines for designing your SAXS experiment. If you have questions, or are a new user, please contact a beamline scientist.
More details about the specifics of BioCAT’s experimental setup can be found elsewhere on our website.
Topics:
What technique should I use?
BioCAT strongly encourages users to use either SEC-SAXS or SEC-MALS-SAXS if possible. If multiple components in solution cannot be separated by size, IEC-SAXS may be the appropriate choice. If components need to be separated by size but are not amenable to size exclusion (such as having strong column interactions or falling apart due to shear or hydrostatic pressure) then AF4-MALS-SAXS is a good choice. This is of particular use for samples like lipid nanoparticles (LNPs).
There are also some cases where sample concentration and volume are inadequate for SEC-SAXS, in which case you will use batch mode. Batch mode is also useful if you need to measure a large number of buffer conditions, such as in a titration experiment, or where your have a precious component in the buffer and cannot provide enough buffer for one of the other methods.
SEC-SAXS is the right choice if …
- Your system has a single well defined peak or several well resolved peaks (not including large aggregates that show up in the void)
- You have a complicated elution with several overlapping or poorly resolved peaks
- You will need to make several buffer changes during your experiment
SEC-MALS-SAXS is the right choice if …
- Your system has a single well defined peak or several well resolved peaks (not including large aggregates that show up in the void)
- You are measuring a complex or oligomer with unknown stoichiometry or molecular weight
- You need a minimal number of buffer changes
- There is little or no elution in the void
IEC-SAXS is the right choice if …
- You have multiple components in solution that cannot be well separated by size
- You can design an IEC gradient that separates your components
AF4-MALS-SAXS is the right choice if …
- Your system is not monodisperse, but cannot be separated well on a size exclusion column due to column interactions or other factors
- Your system is larger than the maximum size that can be separated on a size exclusion column
Batch mode SAXS is the right choice if …
- You can’t meet the concentration and volume requirements for SEC-SAXS (see below)
- You can’t provide enough buffer for SEC-SAXS due to having a precious component (e.g. expensive ligand) in the buffer
- You need a large number (>~5) buffer changes due to the nature of your experiment (e.g. a titration experiment)
- Your system does not survive the a column, for example some weakly associating complexes
What sample concentration and volume do I need?
SEC-SAXS and SEC-MALS-SAXS
As a rule of thumb, you will get good SAXS data if you prepare a protein concentration in mg/ml that is:
- 240/MW in kDa for SEC-SAXS or SEC-MALS-SAXS
For example, for a 20 kDa protein you would want a concentration of ~240/20 = 12 mg/ml whereas for a 150 kDa protein you would want a concentration of ~240/150 = 1.6 mg/ml. RNA and DNA samples scatter ~2.5 times more strongly than protein, so you can use a concentration of ~96/MW.
Note that the MW calculation is done for the expected oligomeric MW. So if a protein is a 50 kDa monomer, but you expect it to be dimeric, you would prepare a concentration of ~240/100 = 2.4 mg/ml.
In SEC-SAXS and SEC-MALS-SAXS experiments the sample may elute in several peaks. This should be accounted for. If a significant amount of your sample elutes in other peaks, further increase your concentration to compensate. Also note that the recommendation above already takes into account the column dilution factor.
Typical SEC-SAXS and SEC-MALS-SAXS injection volumes are ~300 µl. We recommend that you bring enough sample volume for two measurements at each condition, that way if something happens during a measurement (such as the beam going down) we can repeat and obtain the desired data.
Less sample can be used, either as lower concentrations or volumes, either with lower but still decent signal on the same columns or by using the smaller 5/150 columns with less resolution but similar signal. Talk to your beamline scientist about options if you cannot product enough sample.
Batch SAXS
As a rule of thumb, you will get good SAXS data if you target a protein concentration in mg/ml that is:
- 60/MW in kDa
For example, for a 20 kDa protein you would want a concentration of ~60/20 = 3 mg/ml whereas for a 150 kDa protein you would want a concentration of ~60/150 = 0.4 mg/ml. RNA and DNA samples scatter ~2.5 times more strongly than protein, so you can use a concentration of ~24/MW.
Note that the MW calculation is done for the expected oligomeric MW. So if a protein is a 50 kDa monomer, but you expect it to be dimeric, you would prepare a concentration of ~60/100 = 0.6 mg/ml.
For these experiments you need to prepare at least 3 concentrations, ideally bracketing the target concentration in factors of 2 or more. For example, for a 100 kDa protein you might prepare concentrations of 0.6 mg/ml (target), 0.3 mg/ml and 1.2 mg/ml.
Batch mode experiments can be done routinely with ~10 µl per measurement. We recommend that you bring enough sample volume for two measurements at each condition, that way if something happens during a measurement (such as the beam going down) we can repeat and obtain the desired data.
IEC-SAXS and AF4-MALS-SAXS
Concentrations for IEC-SAXS will depending on the loading volume, but you should be aiming for a concentration on elution similar to that of a batch mode experiment. AF4-MALS-SAXS has concentration requirements similar to SEC-SAXS.
IEC-SAXS experiments can have significantly larger loading volumes if desired, and concentration can be done on the column. The Capto HiRes 5/50 have a maximum loading capacity of 50 mg. AF4-MALS-SAXS experiments use sample volumes similar to SEC-SAXS.
In either case, talk to your beamline scientist for more details.
How many samples should I bring?
When considering how many samples to bring, you need to think both about the experiment time and the equilibration.
Experiment time
At BioCAT, users typically use the Superdex 200 Increase 10/300 GL column. This has a column volume of 24 and a flow rates of ~0.6 ml. That means that a 1.25 column volume (CV) experiment for SEC-SAXS or SEC-MALS-SAXS takes ~50 minutes. If you know that nothing elutes after 1 CV (including small molecules) you can further reduce this to ~40 minutes. So you should expect to run ~1-1.5 samples an hour.
With the coflow cell, BioCAT users now have the ability to run samples on the Cytiva 5/150 columns without radiation damage. These columns provide significantly less separation, and so should only be used on a system with well resolved peaks (ideally just one peak, or a peak plus elution at the void volume). However, if your sample is appropriate, the volume requirements and run times are much lower. With these columns, typical load volumes are ~50 µL and run times are ~15 minutes.
Note: If you bring your own column, run times will depend on the flow rate and volume for that column.
The batch mode autosampler can run a sample every ~3 minutes.
IEC-SAXS experiments can take significantly longer, hours to run a single elution depending on the gradient, plus needing an equilibration step after each elution to restore the starting conditions, and so are much lower throughput than SEC-SAXS.
AF4-MALS-SAXS experiments take roughly the same amount of time as a SEC-SAXS experiment.
Equilibration
You will have to equilibrate the column at the start of your SEC-SAXS or SEC-MALS-SAXS experiment, and every time thereafter that you want to change buffers. We recommend a 2 CV equilibration, which for a Superdex 200 Increase 10/300 GL column will take ~1.5 hours.
What column should I use?
BioCAT provides a number of columns for users. Typically, users will use one of these:
- Superdex 75 Increase, both 10/300 and 5/150 (MW ~3-70 kDa)
- Superdex 200 Increase, both 10/300 and 5/150 (MW ~10-600 kDa)
- Superose 6 Increase, both 10/300 and 5/150 (MW ~5-5,000 kDa)
Generally speaking, pick the column with the narrowest MW range that can accommodate your samples. But remember that the MW range is for globular proteins, extended proteins run as if they are higher MW! BioCAT recommends running a test separation in your lab, to ensure you can resolve your species. The default column at BioCAT for all experiments is the Superdex 200 Increase 10/300.
A full list of columns and the corresponding MW ranges is available for both SEC-SAXS and SEC-MALS-SAXS.
We also have standard columns available for IEC-SAXS:
- Capto HiRes Q 5/50
- Capto HiRes S 5/50
Users may also provide their own columns if desired. However, due to the dilution factor, we recommend that you only use analytical grade columns, not the larger prep columns like the Cytiva HiPrep or HiLoad columns.
How much buffer should I bring?
The following are intended as guidelines for users when planning their experiments. However, as most buffers do not contain precious components, we recommend bringing more buffer than you think you’ll need, for example taking the below numbers and adding 50%. You never know when you might want to change buffers and do one more run with a given sample, and have to equilibrate the column again.
If you have precious components in your buffer, there are ways to reduce your buffer usage. We have been able to run SEC-SAXS experiments with as little as ~40 mL of buffer. Please contact a beamline scientist to discuss these situations.
Given the large volume of buffer required for experiments, many of BioCAT’s users find it convenient to bring 10x concentrated stocks of buffer and then dilute on-site.
SEC-SAXS and SEC-MALS-SAXS
For SEC-SAXS experiments, you can calculate the amount of buffer you need as:
Buffer volume = 4*(column volume)*(number of samples + 1) + 250 mL
This accounts for both the per-sample use and the equilibration. Please note that the system cannot use all the buffer in a bottle, as you cannot risk drawing air into the system. There is also a fixed volume used for pump washing. This is the 250 mL offset in the above formula.
For example, if you are using the Superdex 200 Increase 10/300 GL column, it has a column volume of 24 mL. If you’re planning to run 5 samples in a particular buffer you should bring at minimum:
Buffer volume = 4*(24 mL)*(5+1) + 250 mL = 826 mL
As mentioned above, we recommend bringing in excess of this, so for example bring 1.5*826 mL = ~1.2 L of buffer for 5 SEC-SAXS samples with the recommended 50% excess rule.
For these experiments, you should always bring at least 0.5 L of any buffer you are using.
Batch Mode
Batch mode experiments require a basic running buffer that is used for the coflow sheath. This volume should be at least:
Buffer volume = 0.6*(number of samples) + 50 mL
This accounts for both the per-sample use and the equilibration. If you have a standard buffer we recommend bringing significantly more, just for ease of use, ~200 mL should be more than enough for most experiments.
For buffers with precious or limited components, coflow sheath buffer need not contain that component. The same is true if you have lots of buffer changes (e.g. titration of a ligand or salt concentration).
Besides the coflow sheath buffer, you need additional aliquots of a perfectly matched buffer for each sample. If you do not have precious components in the buffer and are not doing lots of buffer changes, we recommend having the coflow sheath buffer be perfectly matched (e.g. via dialysis), as that makes the measurement simple. Note that in this case you should ship the buffer stock at 1x (unlike SEC-SAXS experiments where it’s okay to ship a concentrated buffer for dilution on site).
If you will have mis-matched coflow sheath and sample buffers, you should provide at least:
Buffer volume = 3*(sample volume)
As you typically will bracket the concentration series with buffer measurements. If you wish to make more buffer measurements, e.g. between each sample, then you will need more buffer.
IEC-SAXS and AF4-MALS-SAXS
The required buffer volumes for these experiments depend heavily on the methods used. However, typically these experiments require more buffer than SEC-SAXS experiments. Please consult your beamline scientist for recommendations.