Difference between revisions of "Affinity Purification of Proteins - FPLC"
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==Preparation of Lysates and Buffers== | ==Preparation of Lysates and Buffers== | ||
| − | *Lysates can be prepared by any means, but must be filtered through a 0.22 micron filter prior to applying to the column. | + | *Lysates can be prepared by any means, but must be filtered through a 0.22 micron filter (ie a steriflip) prior to applying to the column. If filtering is slow, do a second high speed spin on the supernatant. |
*Filter all buffers through a 0.22 micron filter. You need an extra ~50 mL of buffer for priming the pumps | *Filter all buffers through a 0.22 micron filter. You need an extra ~50 mL of buffer for priming the pumps | ||
==HPLC Setup== | ==HPLC Setup== | ||
| − | #Check that the HPLC is set up for protein work and not radioactive work (see [[HPLC - Plumbing Setup]]) | + | #Check that the HPLC is set up for protein work and not radioactive work (see [[HPLC - Plumbing Setup]]). |
#Turn on computer, pumps and lamp if not already on by pressing the buttons on the front. | #Turn on computer, pumps and lamp if not already on by pressing the buttons on the front. | ||
| − | #Using TC Navigator, attach the HPLC (if not attached) | + | #Using TC Navigator, attach the HPLC (if not attached). |
| − | #Disconnect the column from the top (if already connected) | + | #Disconnect the column from the top (if already connected). |
#Load the desired method, or write a new one (see [[HPLC - Designing a Method for a Sample or Series]]) | #Load the desired method, or write a new one (see [[HPLC - Designing a Method for a Sample or Series]]) | ||
| − | #Go to setup and | + | #Go to setup and choose where to save your data. |
| − | #Rinse the buffer leads with water and place into the appropriate buffers (see the | + | #Click Hands On and click Start Pump, and on the Detector Tab Click Lamp On (if they are not on already). |
| − | #Prime the pumps in the following order by unscrewing the black wheel in the pump (remove the panel) and sucking up ~25 mL of liquid with a 60 mL syringe: | + | #Rinse the buffer leads with water and place into the appropriate buffers (see the Hands On page for which lead corresponds to which buffer). |
| − | ## | + | #Prime the pumps in the following order by putting the buffer lead into the correct bottle, switching the pump to 100% of that buffer, unscrewing the black wheel in the pump (remove the panel) and sucking up ~25 mL of liquid with a 60 mL syringe: |
| − | ##Elution buffer | + | ##Place the protein lead into the equillibration buffer and prime. |
| − | ##Wash | + | ##Move the protein lead into the protein sample and re-prime with protein (until protein is visible in the syringe). This is done so that the protein sample is only mixed with the equillibration buffer, and this mixture can be added back to the protein sample. |
| − | #Let the equillibration/wash buffer run for a few minutes | + | ##Prime the Elution buffer. |
| − | #Connect the inlet tube to the top of the affinity column (it should still be running wash/equillibration buffer) using the appropriate fittings | + | ##Prime the Wash Buffers and then the Equillibration Buffers. |
| − | #Connect the bottom of the column to the tube leading into the UV monitor | + | #Let the equillibration/wash buffer run for a few minutes. |
| + | #Connect the inlet tube to the top of the affinity column (it should still be running wash/equillibration buffer) using the appropriate fittings. | ||
| + | #Connect the bottom of the column to the tube leading into the UV monitor. | ||
#Check all connections for leaks. | #Check all connections for leaks. | ||
| − | #Set up the Fraction Collector as desired | + | #Set up the Fraction Collector as desired. |
#Check the absorbance (normally 280nm) on the setup panel to ensure that it is ~0. If it is not, let it equillibrate a few more minutes and check it again. If the absorbance is >0.3 then the buffer is absorbing too much light at 280nm. If that is the case, use a method with no UV and turn off the UV lamp from the setup panel. | #Check the absorbance (normally 280nm) on the setup panel to ensure that it is ~0. If it is not, let it equillibrate a few more minutes and check it again. If the absorbance is >0.3 then the buffer is absorbing too much light at 280nm. If that is the case, use a method with no UV and turn off the UV lamp from the setup panel. | ||
#Once everything is ok (and the status panel is marked ready), start the run by turning the injector port to load. If you are loading sample via the pump, either port will work (and can be turned immediately back to the load position). If you are loading via the sample loops, wait until 5 times the loop volume has passed through before turning back. | #Once everything is ok (and the status panel is marked ready), start the run by turning the injector port to load. If you are loading sample via the pump, either port will work (and can be turned immediately back to the load position). If you are loading via the sample loops, wait until 5 times the loop volume has passed through before turning back. | ||
#Check your progress by clicking the real time monitor button. | #Check your progress by clicking the real time monitor button. | ||
| + | #The program should use your entire protein sample plus ~ 10mL. Midway through your run, as the protein starts to run out, add extra equillibration buffer to rinse the tube. | ||
Revision as of 18:04, 13 September 2010
Preparation of Lysates and Buffers
- Lysates can be prepared by any means, but must be filtered through a 0.22 micron filter (ie a steriflip) prior to applying to the column. If filtering is slow, do a second high speed spin on the supernatant.
- Filter all buffers through a 0.22 micron filter. You need an extra ~50 mL of buffer for priming the pumps
HPLC Setup
- Check that the HPLC is set up for protein work and not radioactive work (see HPLC - Plumbing Setup).
- Turn on computer, pumps and lamp if not already on by pressing the buttons on the front.
- Using TC Navigator, attach the HPLC (if not attached).
- Disconnect the column from the top (if already connected).
- Load the desired method, or write a new one (see HPLC - Designing a Method for a Sample or Series)
- Go to setup and choose where to save your data.
- Click Hands On and click Start Pump, and on the Detector Tab Click Lamp On (if they are not on already).
- Rinse the buffer leads with water and place into the appropriate buffers (see the Hands On page for which lead corresponds to which buffer).
- Prime the pumps in the following order by putting the buffer lead into the correct bottle, switching the pump to 100% of that buffer, unscrewing the black wheel in the pump (remove the panel) and sucking up ~25 mL of liquid with a 60 mL syringe:
- Place the protein lead into the equillibration buffer and prime.
- Move the protein lead into the protein sample and re-prime with protein (until protein is visible in the syringe). This is done so that the protein sample is only mixed with the equillibration buffer, and this mixture can be added back to the protein sample.
- Prime the Elution buffer.
- Prime the Wash Buffers and then the Equillibration Buffers.
- Let the equillibration/wash buffer run for a few minutes.
- Connect the inlet tube to the top of the affinity column (it should still be running wash/equillibration buffer) using the appropriate fittings.
- Connect the bottom of the column to the tube leading into the UV monitor.
- Check all connections for leaks.
- Set up the Fraction Collector as desired.
- Check the absorbance (normally 280nm) on the setup panel to ensure that it is ~0. If it is not, let it equillibrate a few more minutes and check it again. If the absorbance is >0.3 then the buffer is absorbing too much light at 280nm. If that is the case, use a method with no UV and turn off the UV lamp from the setup panel.
- Once everything is ok (and the status panel is marked ready), start the run by turning the injector port to load. If you are loading sample via the pump, either port will work (and can be turned immediately back to the load position). If you are loading via the sample loops, wait until 5 times the loop volume has passed through before turning back.
- Check your progress by clicking the real time monitor button.
- The program should use your entire protein sample plus ~ 10mL. Midway through your run, as the protein starts to run out, add extra equillibration buffer to rinse the tube.
