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. BioCAT has a world-leading time-resolved SAXS (TR-SAXS) program that allows users to initiate a reaction through mixing and then measure the changes in the sample as soon as ~45 µs after mixing.
BioCAT provides three different time resolved instruments, which have different accessible time ranges and sample and buffer requirements. These are:
- Chaotic flow mixing, ~45 µs to 60 ms time range
- Laminar flow mixing, ~5 ms to 1.5 s time range
- Stopped flow mixing, >~1 ms time range
Note that longer time points can also easily be accessed via on-plate mixing capabilities of the equilibrium batch mode setup, so talk to a beamline scientist about that possibility if interested.
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 of our continuous flow mixers, the chaotic flow or laminar flow mixing approaches. These will provide the best quality data for your experiments. However, some experiments, particularly if you have a highly viscous buffer or need an extended time range, may be more appropriate for the stopped flow instrument.
Note that both the chaotic flow and laminar flow mixers can be used as part of the same beamtime, and the measured time points can be merged between the two mixers to provide information spanning almost five orders of magnitude in time, from ~45 µs to 1.5 s.
Stopped flow experiments are generally not recommended as they are highly sample and time intensive and are prone to radiation damage.
Chaotic flow mixing is the right choice if …
- You need ultra-fast time points (<1 ms)
- You can provide plenty of sample and buffer
Laminar flow mixing is the right choice if …
- You have minimal sample available
- You want to measure slower time points (>1 ms) or aren’t sure what the time range of interest is
Stopped flow is the right choice if …
- You have a very viscous buffer that isn’t compatible with the continuous flow mixers
- You need time points longer than 1.5 s
What sample concentration and volume do I need?
Chaotic flow mixing
For chaotic flow experiments, we typically recommend a final concentration in mg/ml after dilution that is:
- ~60/(MW in kDa) for protein
- ~24/(MW in kDa) for nucleic acids (due to the higher contrast)
Note that in chaotic mixing experiments the sample undergoes typically between 4-10x dilution.
For example, to measure a 100 kDa protein we would recommend a concentration after dilution of 0.6 mg/ml. For example, to measure a 100 kDa protein we would recommend a concentration after dilution of 0.6 mg/ml. At a typical dilution factor of 5x, this would be a starting concentration of 3.0 mg/ml. Whereas for a 100 kDa nucleic acid, we would recommend a diluted concentration of 0.24 mg/ml, and so a starting concentration (for 5x dilution) of 1.2 mg/ml.
With this mixer we typically measure the time series using a 1 mL injection, and make 3 repeated measurements to improve signal to noise, for a total loaded volume of 3 mL.
Note that with these concentrations and volumes, a chaotic flow experiment with a 100 kDa protein would take less than 10 mg for the entire time series
Laminar flow mixing
For laminar flow experiments, we typically recommend a concentration in mg/ml of:
- ~120/(MW in kDa) for proteins
- ~48/(MW in kDa) for nucleic acids (due to the higher contrast)
For example, a 100 kDa protein would be injected at a concentration of ~1.2 mg/ml and a 100 kDa RNA would be injected at a concentration of ~0.48 mg/ml.
With this mixer, we typically measure the time series using a 100 µL injection, and make 3 repeated measurements to improve signal to noise, for a total loaded volume of 300 µL.
Note that these recommended concentrations are 2x lower and the loading volume is equivalent to what we recommend for SEC-SAXS experiments, so a laminar flow time series can be measured with less material than a single SEC-SAXS injection. For many proteins and nucleic acids these experiments require significantly less than 1 mg of material, making them feasible for essentially every system that can be measured by equilibrium SAXS.
How many samples should I bring?
Below we give recommendations for how many samples to bring in your beamtime. However, time-resolved SAXS experiments are often more exploratory than equilibrium measurements, as the exact time range over which the behavior occurs may not be known, and so often more time is spent analyzing and iterating on conditions and measured time ranges than is spent measuring data.
Chaotic flow mixing
A typical chaotic flow experiment will run for ~10 minutes in order to collect pre and post buffer measurements as well as the sample measurement. It takes ~5 minutes to load a sample and start data collection. A final time series usually consists three repeated measurements of the same sample, so you can plan on measuring roughly one time series per hour.
If you need to change buffers between that takes roughly 30 minutes.
Laminar flow mixing
A typical laminar flow experiment will run for ~30 minutes in order to collect pre and post buffer measurements as well as the sample measurement. It takes ~5 minutes to load a sample and start data collection. A final time series usually consists three repeated measurements of the same sample, so you can plan on measuring roughly one time series per two hour.
If you need to change buffers between that takes roughly 60 minutes.
What buffers should I use?
Sample and buffer preparation is done in the standard way for SAXS experiments. Because there is no in-line purification (such as for SEC-SAXS), the samples must be of sufficient monodispersity and homogeneity that you could make batch mode SAXS measurements with them.
Note that we have two distinct buffers for mixing experiments. The first is the “sample buffer”, which is the initial state the sample is in. The second is the “mixing buffer”, which is the buffer that will be mixed into the sample buffer to create the final buffer. The excess sample buffer is used to inject the sample and to make the necessary buffer measurements for the experiment, so it must be perfectly matched to the actual buffer the sample is in (e.g. by dialysis).
For these experiments we recommend including a radical scavenger to protect against radiation damage. Typically we recommend 1-2% glycerol, but in some cases reductant (e.g. DTT or EDTA) or other radioprotectants may be used.
Chaotic flow mixing
Buffers should be prepared taking into account final dilution ratios of the mixing. The chaotic flow mixer is usually run at sample high dilution, typically between 4-10x. For example, at the 5x dilution, any components in the sample buffer that are not in the mixing buffer will be diluted 5x, and any components in the mixing buffer that are not in the sample buffer will be diluted by 4/5th.
You can use the following formula (where IC standards for initial concentration, DR for dilution ratio, SB sample buffer, and MB mixing buffer) to calculate any parameter if you fix the others. For example, you could calculate the mixing buffer concentration given a target final value, initial value, and dilution ratio:
- Final concentration = (1 - 1/(DR))*(IC in MB) + (1/DR)*(IC in SB)
Note that if you have zero of the component in either buffer this formula simplifies.
Suppose you want to do a salt dilution experiment, where the sample goes from high to low salt. If you know the sample needs to start in 1 M start for the initial state of interest and you want a mixed state with 150 mM salt, you would then pick a dilution ratio of 1000 mM/150 mM = 6.67, using a zero salt mixing buffer. On the other hand, if you wanted a final state of 150 mM salt but didn’t care what the starting concentration was as long as it was above 500 mM, you could stick with the standard dilution ratio of 5x and use a starting concentration of (150 mM)*5 = 750 mM, again with a zero salt mixing buffer.
Equivalently, a salt jump experiment mixing from a zero salt sample buffer into a high salt mixing buffer is easy to calculate. If you want a final salt condition of 500 mM, and you are using a dilution ratio of 5x, the necessary starting concentration of the mixing buffer is (500 mM)/(1-1/5)) = 625 mM.
For the more complicated case, consider a salt jump experiment where you start with low but non-zero salt and do a 5x dilution into a high salt buffer. If you start with the sample in a 50 mM salt in the sample buffer, and you wanted to end with 500 mM salt, you would need to have a mixing buffer with a salt concentration of:
- (Final concentration - (IC in SB)/DR)/(DR-1/DR) = (IC in MB)
- (500 mM - (50 mM)/5)/(4/5) = 612.5 mM
Note that any component can be changed via mixing, salt is just a sample example. If you are changing pH, note that the above formulas will not apply.
Components for which you do not want to change the concentration should be at the same concentration in both sample and mixing buffers.
Laminar flow mixing
In the laminar flow mixer the dilution is fixed by the ratios of the flow rate of the sample buffer and mixing buffer. Components in the sample buffer will get diluted to 8.7% (11.5x) of their initial value and components in the mixing buffer will get diluted to 91.3% (1.1x) of their initial value. You can calculate the final mixed concentration as (where IC is initial concentration, SB is sample buffer and MB is mixing buffer)
- Final concentration = 0.087*(IC in SB) + 0.913*(IC in MB)
Consider the example of mixing from high salt in the sample buffer to low salt in the final buffer. If the salt in the mixing buffer is zero, then the final concentration = 0.087*(initial concentration in sample buffer). So if you want a final concentration of 150 mM, you’d need an initial concentration of (150 mM)/0.087 = 1.724 M. Alternatively, if you wanted to start with a 500 mM solution, you would achieve a final concentration of (500 mM)*0.087 = 43.5 mM.
Equivalently, consider a salt jump experiment mixing from a zero salt sample buffer to a high salt final buffer. If you want a final salt concentration of 500 mM, you would use a mixing buffer with (500 mM)/0.913 = 548 mM salt.
For a more complicated sample, consider a case where you want to start with 50 mM salt in the sample buffer and end with 500 mM salt final buffer. The necessary mixing buffer salt concentration would be:
- (Final concentration - (0.087*(IC in SB)))/0.913 = IC in MB
- (500 mM - (0.087*(50 mM)))/0.913 = 543 mM
Note that any component can be changed via mixing, salt is just a sample example. If you are changing pH, note that the above formulas will not apply.
Components for which you do not want to change the concentration should be at the same concentration in both sample and mixing buffers.
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 do a couple more measurements with a given sample.
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. This can only be done for the buffer the sample is mixing into, the sample running buffer must be perfectly matched to the buffer the sample is in, such as with dialysis.
Chaotic flow mixing
Chaotic flow experiments involve dilution of the sample into the mixing buffer. Dilution factors can be whatever the user desires, but typically are between ~4-10x, and 5x is considered standard. Flow rates can also be varied to adjust the time range measured and are typically between 4.5-8 mL/min, with 6 mL/min being standard.
You can calculate the necessary buffer as:
- Sample buffer = (10 min)*(flow rate)*(number of measurements+1)*(1/(dilution ratio)) + 50 mL
- Mixing buffer = (10 min)*(flow rate)*(number of measurements+1)*(1 - 1/(dilution ratio)) + 50 mL
So, for example, for the standard flow rate of 6 mL/min and the standard dilution factor of 5x, to measure a time series consisting of 3 repeated measurements on the same sample, you would prepare:
- Sample buffer = (10 min)*(6 mL/min)*(4)*(1/5) + 50 mL = 98 mL
- Mixing buffer = (10 min)*(6 mL/min)*(4)*(4/5) + 50 mL = 242 mL
Talk to your beamline scientist for recommendations on flow rate and dilution factor.
Laminar flow mixing
Laminar flow experiments are typically run at fixed ratios between the mixing buffer and sample buffer and at standard flow rates. You can calculate the necessary buffer as:
- Sample buffer = (0.4 mL)*(number of measurements) + 10 mL
- Mixing buffer = (4.1 mL)*(number of measurements) + 20 mL
So, for example, to measure a time series consisting of 3 repeated measurements on the same sample, you would prepare:
- Sample buffer = (0.4 mL)*3 + 10 mL = 11.2 mL
- Mixing buffer = (4.1 mL)*3 + 20 mL = 32.3 mL