Difference between revisions of "Surface Plasmon Resonance - Protein Lipid Interactions"
From Bridges Lab Protocols
m (Additions from protocol handwritten by Dave.) |
Davebridges (Talk | contribs) (updated protocol to make 300 uL of lipid to make extruding easier) |
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| + | __NOTOC__ | ||
==Materials== | ==Materials== | ||
*L1 Sensor Chip (Biacore) | *L1 Sensor Chip (Biacore) | ||
| − | *Dioleylphosphatidylcholine (Sigma) dissolved in 1:1 chloroform:methanol with 0.1% HCl | + | *10x Dioleylphosphatidylcholine (Sigma) dissolved in 1:1 chloroform:methanol with 0.1% HCl |
| − | *PIPx of interest (Avanti or Echelon) dissolved in 1:1 chloroform:methanol with 0.1% HCl | + | *10x PIPx of interest (Avanti or Echelon) dissolved in 1:1 chloroform:methanol with 0.1% HCl |
| − | *HBS-N (25mM HEPES, pH 7.4, 150 mM NaCl)(6.25mL Hepes (1M), 9.38mL NaCl (4M)) Total volume 250mL. | + | *HBS-N (25mM HEPES, pH 7.4, 150 mM NaCl)(6.25mL Hepes (1M), 9.38mL NaCl (4M)) Total volume 250mL. Sterile filter. |
| − | *1M NaOH | + | *1M NaOH. |
*pH strips | *pH strips | ||
| − | *Water bath sonicator | + | *Water bath sonicator warmed to 40C |
*Avanti MiniExtruder | *Avanti MiniExtruder | ||
| − | *1% beta octylglucoside | + | *1% beta octylglucoside. Sterile filter or centrifuge for 10 min at 13 000 RPM. |
| − | *0.5% SDS | + | *0.5% SDS. Sterile filter or centrifuge for 10 min at 13 000 RPM. |
| − | *30% ethanol | + | *30% ethanol. Sterile filter or centrifuge for 10 min at 13 000 RPM. |
| − | *100mM NaOH | + | *100mM NaOH. Sterile filter or centrifuge for 10 min at 13 000 RPM. |
==Preparation of Liposomes== | ==Preparation of Liposomes== | ||
| − | #Combine DOPC + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen ( | + | #Combine DOPC + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (30 uL PC or 29.1 uL PC + 9 uL PI) |
| − | #Resuspend to 10 mM total lipid with HBS-N ( | + | #Resuspend to 10 mM total lipid with HBS-N (300 uL). Vortex thoroughly and sonicate in water bath |
| − | #Correct pH to 7.4 using pH paper and | + | #Correct pH to 7.4 using pH paper and (usually ~ 1uL of 1M NaOH) |
#Freeze thaw in a water bath sonicator at 40C and liquid nitrogen 8 times. | #Freeze thaw in a water bath sonicator at 40C and liquid nitrogen 8 times. | ||
#Pass 10X through a polycarbonate filter using an Avanti MiniExtruder at Room Temperature. | #Pass 10X through a polycarbonate filter using an Avanti MiniExtruder at Room Temperature. | ||
| Line 23: | Line 24: | ||
*Place PC filter on block. | *Place PC filter on block. | ||
*Tightly attach syringe and place on blcok. | *Tightly attach syringe and place on blcok. | ||
| − | *Pass 250uL of milliQ water through the | + | *Pass 250uL of milliQ water through the filter ~5 times. |
| − | *Discard the water and inject the sample through the filter 10 times. | + | *Discard the water and inject the sample through the filter 10 times. |
| − | + | ||
==Preparation of Surface== | ==Preparation of Surface== | ||
| − | #Wash all four surfaces at 10 uL/min and 25C with HBS-N | + | #Aliquot ~200 uL of beta-octylglucoside, SDS and ethanol solutions into plastic vials and label. Tap to make sure there are no bubbles at the bottom of the vial. |
| − | #Inject 20 uL of 1% beta-octylglucoside | + | #Aliquot the lipid solutions into plastic vials and label. Providing no sample leakage occured, you should have close to 100 uL of sample. The injection will require exactly 100 uL of sample so check the volume by pipetting, and add HBS to increase the volume to >110 uL. |
| − | #Inject 20 uL of 0.5% SDS | + | #Copy the '''Lipid Loading''' program to a new folder for the day's experiments. |
| − | #Inject 10 uL of 1% beta-octylglucoside | + | #Edit the program by entering after the '''!''' which lipid is loaded on each surface. Start with lane 4 and move to lane 1-PC only, lipids can migrate accross wells over time, so load in a way to minimize contamination. For example, if possible PC should precede the lipid of interest on the surface. |
| − | #Inject 10 uL of 30% ethanol | + | #Place the beta-octylglucoside, SDS, ethanol and lipid solutions in the appropriate spot in the block (Remember that R2 is on the right, and position 1 is closest to you). |
| − | #Inject 70 uL of extruded lipid suspension at 10uL/min (should see an increase of 5000-10000RU) to desired | + | #Save the program with the current date. |
| − | #Wash with 20 uL of 0.1M NaOH | + | #From the Biacore Control Software enter Run Method, then select the method you just created. If there is a syntax error you will need to edit the program to fix it. The program will do the following injections: |
| + | ##Wash all four surfaces at 10 uL/min and 25C with HBS-N. | ||
| + | ##Inject 20 uL of 1% beta-octylglucoside. | ||
| + | ##Inject 20 uL of 0.5% SDS. | ||
| + | ##Inject 10 uL of 1% beta-octylglucoside. | ||
| + | ##Inject 10 uL of 30% ethanol. | ||
| + | ##Inject 70 uL of extruded lipid suspension at 10uL/min (should see an increase of 5000-10000RU) to desired surface. | ||
| + | ##Repeat previous step for all lipids. | ||
| + | ##Wash all surfaces with 20 uL of 0.1M NaOH. | ||
| + | |||
==Analysis of Sample== | ==Analysis of Sample== | ||
| − | #Inject protein samples adjusting contact time as necessary to reach saturation | + | #Ideally, you would inject a HBS buffer blank, followed by increasing, then decreasing concentrations of your samples. For a kinject, the volume required is the injection volume + 40 uL so ensure that you have at least that much plus 10 uL per injection to avoid injecting air. |
| − | #Inject 20 uL 0.1M NaOH between samples | + | #Inject protein samples adjusting contact time as necessary to reach saturation. Typically we use a 400s contact time (66 uL at 10 uL/min). It is important that at higher concentrations an equillibrium is reached, if equillibrium is reached early consider reducing this time. If the curve is not flat on the top, consider increasing the contact time. Adjust the contact time by manipulating the volume injected (not the flow rate). Set the dissociation portion of a kinject to be 100s. |
| − | #For Kd determination, | + | #Inject 20 uL 0.1M NaOH between samples. If you have very low binding levels (less than 50 RU), use a kinject, with a 600s dissociation to ensure that the surface is totally re-equillibrated. Otherwise use a straight inject. |
| + | #For Kd determination, do a pilot series, calculate the approximate Kd then redo the titration with 5 concentrations above and below the kd in 2x intervals. | ||
| + | #To run sample, edit an existing injection program with the above considerations and then save in your folder for the day. | ||
| + | #Run this program using the Biacore Control Software and Run Method. | ||
| + | #Analyse the data using [[Using Scrubber2 | Scrubber2]] and/or a separate analysis graphing package such as [[Using R to Determine Ligand Binding Constants | R ]], SigmaPlot or Graphpad. | ||
==Reference== | ==Reference== | ||
| − | + | PMID 16829131 | |
| + | [[Category: Lipid Analysis]] | ||
| + | [[Category: Inositol Lipids]] | ||
| + | [[Category: Protein-Lipid Interactions]] | ||
Latest revision as of 14:40, 14 April 2011
Materials
- L1 Sensor Chip (Biacore)
- 10x Dioleylphosphatidylcholine (Sigma) dissolved in 1:1 chloroform:methanol with 0.1% HCl
- 10x PIPx of interest (Avanti or Echelon) dissolved in 1:1 chloroform:methanol with 0.1% HCl
- HBS-N (25mM HEPES, pH 7.4, 150 mM NaCl)(6.25mL Hepes (1M), 9.38mL NaCl (4M)) Total volume 250mL. Sterile filter.
- 1M NaOH.
- pH strips
- Water bath sonicator warmed to 40C
- Avanti MiniExtruder
- 1% beta octylglucoside. Sterile filter or centrifuge for 10 min at 13 000 RPM.
- 0.5% SDS. Sterile filter or centrifuge for 10 min at 13 000 RPM.
- 30% ethanol. Sterile filter or centrifuge for 10 min at 13 000 RPM.
- 100mM NaOH. Sterile filter or centrifuge for 10 min at 13 000 RPM.
Preparation of Liposomes
- Combine DOPC + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (30 uL PC or 29.1 uL PC + 9 uL PI)
- Resuspend to 10 mM total lipid with HBS-N (300 uL). Vortex thoroughly and sonicate in water bath
- Correct pH to 7.4 using pH paper and (usually ~ 1uL of 1M NaOH)
- Freeze thaw in a water bath sonicator at 40C and liquid nitrogen 8 times.
- Pass 10X through a polycarbonate filter using an Avanti MiniExtruder at Room Temperature.
- Rinse syringe with milliQ water.
- Wet filter paper and put the filter on the block.
- Place PC filter on block.
- Tightly attach syringe and place on blcok.
- Pass 250uL of milliQ water through the filter ~5 times.
- Discard the water and inject the sample through the filter 10 times.
Preparation of Surface
- Aliquot ~200 uL of beta-octylglucoside, SDS and ethanol solutions into plastic vials and label. Tap to make sure there are no bubbles at the bottom of the vial.
- Aliquot the lipid solutions into plastic vials and label. Providing no sample leakage occured, you should have close to 100 uL of sample. The injection will require exactly 100 uL of sample so check the volume by pipetting, and add HBS to increase the volume to >110 uL.
- Copy the Lipid Loading program to a new folder for the day's experiments.
- Edit the program by entering after the ! which lipid is loaded on each surface. Start with lane 4 and move to lane 1-PC only, lipids can migrate accross wells over time, so load in a way to minimize contamination. For example, if possible PC should precede the lipid of interest on the surface.
- Place the beta-octylglucoside, SDS, ethanol and lipid solutions in the appropriate spot in the block (Remember that R2 is on the right, and position 1 is closest to you).
- Save the program with the current date.
- From the Biacore Control Software enter Run Method, then select the method you just created. If there is a syntax error you will need to edit the program to fix it. The program will do the following injections:
- Wash all four surfaces at 10 uL/min and 25C with HBS-N.
- Inject 20 uL of 1% beta-octylglucoside.
- Inject 20 uL of 0.5% SDS.
- Inject 10 uL of 1% beta-octylglucoside.
- Inject 10 uL of 30% ethanol.
- Inject 70 uL of extruded lipid suspension at 10uL/min (should see an increase of 5000-10000RU) to desired surface.
- Repeat previous step for all lipids.
- Wash all surfaces with 20 uL of 0.1M NaOH.
Analysis of Sample
- Ideally, you would inject a HBS buffer blank, followed by increasing, then decreasing concentrations of your samples. For a kinject, the volume required is the injection volume + 40 uL so ensure that you have at least that much plus 10 uL per injection to avoid injecting air.
- Inject protein samples adjusting contact time as necessary to reach saturation. Typically we use a 400s contact time (66 uL at 10 uL/min). It is important that at higher concentrations an equillibrium is reached, if equillibrium is reached early consider reducing this time. If the curve is not flat on the top, consider increasing the contact time. Adjust the contact time by manipulating the volume injected (not the flow rate). Set the dissociation portion of a kinject to be 100s.
- Inject 20 uL 0.1M NaOH between samples. If you have very low binding levels (less than 50 RU), use a kinject, with a 600s dissociation to ensure that the surface is totally re-equillibrated. Otherwise use a straight inject.
- For Kd determination, do a pilot series, calculate the approximate Kd then redo the titration with 5 concentrations above and below the kd in 2x intervals.
- To run sample, edit an existing injection program with the above considerations and then save in your folder for the day.
- Run this program using the Biacore Control Software and Run Method.
- Analyse the data using Scrubber2 and/or a separate analysis graphing package such as R , SigmaPlot or Graphpad.
