This provides instructions of how to collect SEC-SAXS data at the BioCAT beamline.Before comming, please ensure that you’ve read up on how toplan your experimentand that you’ve properly prepared your samples.
Contents
- Before datacollection
- Changing buffer
- Changing buffer on the AKTA
- Changing coflow sheathbuffer
- LoadingSample
- Set up data collection
- Setting a method for the AKTA
- Setting exposureparameters
- Start datacollection
- End datacollection
- Optimizing yourtime
Before datacollection
There are a few commonprocesses:
- Every buffer you use for SEC-SAXS should be filtered anddegassed.
- In SEC-SAXS experiments you should reserve ~50 mL of buffer in a falcontube. This will be used for cleaning the sample loop, diluting samples,etc. It is convenient to not have to extract this from the larger bufferbottles.
- Every sample should be spun down for 10 minutes just beforemeasurement.
Changingbuffer
To change buffer you must do two things. First, you need to equilibrate thecolumn in the new buffer. Second, you need to change the coflowbuffer.
IMPORTANT: Never change directly from a buffer with salt to a storage solutionof 20% ethanol, or vice versa. This could cause salt to crash out of solutionand damage equipment. Always include a water equilibration step between thosetypes ofsolutions.
Changing buffer on the AKTA
IMPORTANT: A beamline scientist should train you on how to change bufferbefore you do it on yourown.
SEC-SAXS uses the BioCAT AKTA Pure FPLC, which is controlled by UNICORN 6.To change buffer you need to equilibrate the column you will use. To doso:
In the UNICORN 6 control software, stop any ongoing run by clicking the stopbutton.
(Video) SEC-SAXS data processing tutorial
Uncap the new buffer bottle. Remove the A and B frits from the current buffer.Over a waste beaker, rinse both frits and tubing with water from a DI Watersquirtbottle.
- IMPORTANT: You may be using the same buffer bottle for the FPLC as thecoflow sheath. If so, make sure to stop the FPLC before transferring theFPLC leads with the coflowleads.
Place A and B frits into the new buffer. If necessary, cut and stretch a piece of parafilmover the bottle top to prevent contaminants such as dust entering the bottle.Cap the old bufferbottle.
If it is not open, open the manual instructions window by selecting theManual->Execute Manual Instructions menuoption.
Run a pumpwash.
- In the instructions panel of the Manual instructions window, expand pumpsand click on “Pump Awash”.
- In the Parameters section select inletA1.
- In the instruction execution section clickInsert.
- Do the same except for Pump B, selecting inletB1.
- Click the Execute button to start a pumpwash.
Set the columnposition.
- Expand the Flow path section and click on “Columnposition”.
- Select the appropriate position for your column (you can trace tubingfrom the column valve). If you’re unsure, ask a beamlinescientist.
- Make sure flow direction is downflow and clickExecute.
Set a pressure alarm. Just below the column pressure limit, usually 2.8MPa.
- Expand the Alarms section and click on “Alarm delta columnpressure”.
- Set mode toEnabled.
- Set high alarm to just below the column pressure limit. For the GEincrease columns set the alarm to 2.8 MPa. For the GE non-increasecolumns set the alarm to 1.4MPa.
- ClickExecute.
Start flow. Flow rate usually 0.7mL/min
- In the Pumps section, flick on System flow. Enter the flow rate,usually 0.7mL/min.
- ClickExecute.
Equilibration is now running. You should equilibrate for 2 column volumns.Check periodically to verify that the high alarm has not tripped. If thishappens you need to adjust the flow rate down to reduce the pressure. Thisshould only be an issue if buffers have a high concentration of glycerol,if it occurs otherwise it may indicate a problem with the column. In thatcase contact a beamlinescientist.
You will also need to change the coflow sheathbuffer.
Changing coflow sheathbuffer
IMPORTANT: A beamline scientist should train you on how to change bufferbefore you do it on yourown.
Whenever the buffer is changed for a SEC-SAXS or SEC-MALS-SAXS experiment thecoflow sheath buffer also need to be changed. To doso:
- In the experiment control software, stop the coflow by clicking the “Stop Coflow”button.
- Uncap the new buffer. Remove the coflow buffer tubing from the currentbuffer.
- Remove the pierced cap from the current buffer and place it on the new buffer.If there are any drops of the old buffer on it, gently with the cap with a kimwipe.
- IMPORTANT: You may be using the same buffer bottle for the FPLC as thecoflow sheath. If so, make sure to stop the FPLC before transferring theFPLC leads with the coflowleads.
- Over a waste beaker, rinse the tubing with water from a DI Watersquirtbottle.
- Place the coflow buffer line in the newbuffer.
- In the experiment control software, start the coflow by clicking the “Start Coflow”button.
The coflow sheath flow should be given ~10 minutes to equilibrate. If you are low onbuffer or doing a SEC-MALS-SAXS equilibration you can can then stop the sheathflow while the rest of the system equilibrates. If you are doing a SEC-SAXS equilibrationand have plenty of buffer, BioCAT recommends leaving the sheath flow runningso that you can’t forget to start it for yourexperiment.
LoadingSample
IMPORTANT: A beamline scientist should train you on how to load samplebefore you do it on yourown.
Immediately before loading a sample you should spin down the sample for 10minutes.
To loadsample:
In the UNICORN 6 control software, stop any ongoing run by clicking the stopbutton.
If it is not open, open the manual instructions window by selecting theManual->Execute Manual Instructions menuoption.
Set the loop valveposition.
- In the instructions panel of the Manual instructions window, expand“Flow path” and click on “Loopvalve”.
- In the Parameters section select the appropriate position. You can verifythe position by looking at the loop valve on the AKTA. If you’re unsureof the position ask as beamlinescientist.
- ClickExecute.
- WARNING: If you select the wrong loop, or leave the valve in bypass,your sample could be lost! If you have any question about this ask abeamlinescientist.
Set the injection valveposition.
- In the “Flow path” section select “Injectionvalve”.
- Set the position to “ManualLoad”.
- ClickExecute.
- WARNING: If you select the wrong injection valve position,your sample could be lost! If you have any question about this ask abeamlinescientist.
Flush the loop. Use a total of 5x loop volume when changing buffers, 2xloop volume between samples in the samebuffer.
- Fill a syringe with buffer to more than the selected loop’s volume.Remove the needle used for filling (ifany).
- Put the syringe in the load port on the AKTA.
- Empty the entire syringe volume through theloop.
- Repeat once for a new sample in the same buffer. Repeat 5 timesif you are changingbuffers.
- IMPORTANT: Leave the syringe in the load port after the finalflush
- If you want to clean the loop, rather than just flush, talk to a beamlinescientist. For most samples this is notnecessary.
Load thesample.
- Fill a syringe with the sample loading volume. Remove the needle used forfilling (ifany).
- Invert the syringe (tip up) and tap to drive any air bubbles to thetop.
- With the syringe inverted, push the plunger until the sample is all the wayto the tip leaving no air in thesyringe.
- Remove the empty buffer syringe from the load port and immediately placethe sample syringe in theport.
- IMPORTANT: If you wait after removing the buffer syringe, some volumemay siphon out of the loop, letting air enter thesystem.
- Empty the syringe into theloop.
- IMPORTANT: Leave the syringe in the loadport.
Set up datacollection
Setting a method for the AKTA
IMPORTANT: A beamline scientist should train you on how to set up an AKTA methodbefore you do it on yourown.
To set up a method for arun:
In the UNICORN 6 control software, stop any ongoing run by clicking the stopbutton.
If available, click the “Run”button.
If the run button is grayed out, click the ‘Open Method Navigator’ button.Then double click the sup2005150method.
(Video) BioXTAS RAW - Basic SEC-SAXS analysisA “Start Protocol” window will open. We will only refer to items within that window goingforward.
In the variable list check and change asappropriate:
- Columntype
- Columnposition
- Flow rate (default 0.7 ml/min for 10/300 increasecolumns)
- Loop position (NOT Sample LoopPosition)
- Empty loop with (should be at least 2 loopvolumes)
- Isocratic elution length (should be1.5)
- If you aren’t sure what any of these variables should be, contact yourbeamlinescientist.
- Note that you may have to scroll down in the list to find some of thesevariables.
Click the “Next” button three times to advance to the “Method Information” screen.In the “Method Duration” tab take note of the totaltime.
Click the “Next” button three times to advance to the “Result Name and Location”screen.
Select the appropriate directory (should be /DefaultHome/<run>/<user>, such as/DefaultHome/2019-1/20190430Hopkins for the first run of 2019, and a user groupwith PI Hopkins on 04/30/2019). The first time you run a method you will have tocreate thisfolder.
Give the run a identifiable name. The BioCAT default is PI initials plussample number (starting at 1 and incrementing with each sample, for exampleJH1 for the first sample of a group with PI with initials JH).
You are now ready to start the method. You shouldn’t start it until you’ve closedthe hutch and set the proper exposure parameters, so leave the “Start Protocol”window open fornow.
Setting exposureparameters
IMPORTANT: A beamline scientist should train you on how to set exposureparameters before you do it on yourown.
To set exposure parameters in the BioCAT controlsoftware:
Make a new folder for your sample by clicking on the folder button.It will be contained within your top level directory (should match allother top level directory names, such as 2019043_Hopkins for a usergroup with PI Hopkins on 04/30/2019). Name the folder consistent withthe sample identification in the FPLC/HPLCmethod.
- The BioCAT default for a sample name is PI initials plus sample number(starting at 1 and incrementing with each sample, for exampleJH1 for the first sample of a group with PI with initials JH).
Change the filename to the new sample name. This should be consistent withthe folder name and with the sample identification in the FPLC/HPLCmethods.
- The BioCAT default for a sample name is PI initials plus sample number(starting at 1 and incrementing with each sample, for exampleJH1 for the first sample of a group with PI with initials JH).
Set the exposure time and exposure period appropriately. The defaults thatmost users will want to use are 0.5 s and 1 s for time and periodrespectively.
- Note: You will usually not need to change this. Check anyways just toto besure.
Set the number of frames appropriately. The default most users will want touse is 3600. verify that frames*exposure period is equal to or greater thanthe run time of your FPLC/HPLCmethod.
Set the “LC Flow Rate” to the flow rate of your method. If coflow is onclick the “Change Flow Rate”button.
(Video) BioXTAS RAW - Baseline correction for SEC-SAXS data- Note: You will usually not need to changethis.
If coflow is off click the “Start Coflow”button.
If you’re not sure what any of the above parameters should be, contact yourbeamlinescientist.
Your exposure parameters are now set. You’re ready to start your datacollection.
Start datacollection
Starting data collection is nowsimple.
- First start the AKTA method by clicking the “Start” button in the“Start
- Protocol”window.
Then wait until a predetermined time and click the “Start Exposure” button.How long you wait depends on the column you are using, but generally speakingyou should start the exposure just after the sample is injected. Talk toyour beamline scientist for more guidance with your particularexperiment.
At this point you should also start on-line processing of the SAXSdata.
Monitor the progress of the elution and the SAXS data to ensure nothing unexpectedoccurs during yourrun.
End datacollection
The data collection will naturally end when your FPLC method ends and whenyour exposures end. If you are certain that you have collected all of thedata (i.e. everything of interest has eluted and passed through the SAXS celland the SAXS intensity has returned to baseline) you can end your datacollection early. To do this, press the “Stop Exposure” button in the exposurecontrolsoftware.
If everything has eluted from the injection (including any salt or other smallmolecules) you can also stop the FPLC method. Only do this if you arecertain that everything has eluted, otherwise let it run the full 1-1.5 CV.
To do so press the “Stop” button the AKTA controlsoftware.
Optimizing yourtime
There are several things to keep in mind to help you optimize yourtime:
- Buffer changes on either instrument, but particularly the SEC-MALS-SAXS,take a lot of time. Optimize by combining samples into the same bufferas much as possible. Also make sure you know what experiments you’re doingin which buffer and do them all at once so you don’t have tore-equilibrate.
- If you are doing both SEC-SAXS and SEC-MALS-SAXS, you can do one or the otherwhile equilibrating the other system. A typical sequence might be:
- Equilibrate one or both of the SEC-MALS-SAXS systemsovernight.
- In the morning at the start of your beamtime start to equilibrate theSEC-SAXSsystem.
- Collect data on one or both of the SEC-MALS-SAXSsystems.
- Start those systemsequilibrating.
- Switch to the SEC-SAXS system and runsamples.
- Switch back to the SEC-MALS-SAXSsystems.
- Groups with a lot of buffer changes can pre-equilibrate columns off-lineon our preparative FPLC while running experiments on the AKTA.
- You should start spinning down your next sample with ~10-15 minutes leftin your current run. This means starting to prepare any dilutions necessaryas soon as you’ve started data collection on your currentsample.
- If you’re sure all of the injection, including small molecules has eluted,you can stop your data collection early. Many users are able to stop datacollection after 1 CV, and don’t need the entire 1.5 CV elution to clearthecolumn.
- If you are using the SEC-MALS-SAXS instrument, once you have stopped the SAXSdata collection you can load your next sample into the autosampler withoutwaiting for the HPLC run tofinish.