For the cross-linking, followed by immunoprecipitation. Procedure cells at 80 to 90%density are incubated with one millimolar ice cold. DSP in PBS, calcium, magnesium, or DMSO only.
Vehicle control. DSP is a homo bifunctional membrane permeable and th reducible crosslinker that crosslinks free and means within 12 angstroms of each other. Following cross-linking cells are lysed and immuno precipitations are performed using standard controls.
Protein complexes are then resolved by reducing SDS page, reducing agents'cleaved DSP, allowing for the identification of individual proteins by immuno blot. Hi, I'm Stephanie Ick from the laboratory of Dr.Victor Fonde in the Department of Cell Biology at Emory University. And I'm Pearl Ryder, also from the Fonde lab.
Today we'll be showing you a procedure for the isolation of myprotein complexes using controlled cross-linking followed by immuno magnetic affinity chromatography. We use this procedure in our laboratory to study transient labile protein interactions. So let's get started.
Begin by labeling the bottom of the cell plates to indicate the cell type and specific experimental condition. A positive symbol here indicates DSP treated include a negative DMSO only vehicle control. Add media to the plates to prepare them for cell seeding plate.
HEK cells seeded at a one to five dilution to allow isolation of 500 micrograms of protein per standard tube reaction. A single tube may suffice to identify the putative binding partners of a protein of interest by immuno blot from mass spectrometry analysis. Increase the number of standard reactions at least 10 times.
Also prepare stalks of most of the solutions used in the cross-linking experiment. These include PBS supplemented with 0.1 millimolar calcium chloride and one millimolar magnesium chloride termed PBS calcium magnesium. The addition of calcium and magnesium ions is critical for adhesion of cells to the culture plate.
During the course of the experiment, store the solution at four degree Celsius. Other stocks include crosslinker quenching solution 50 X complete protease inhibitor cocktail, which is vortex every 20 minutes until the pellet is dissolved. 20%Tritan X 100, which is stored at four degrees Celsius and should be used within one month and 10 x and one x Buffer a solutions.
Prepare and store all the solutions as detailed in the accompanying written protocol. Finally, prepare the lysis buffer made up of one X buffer. A plus 0.5%TRITTON X 100 and immuno magnetic precipitation buffer or IP buffer made up of one X buffer.
A plus 0.1%TRITTON X 101 to two days after plating, the cells are 80 to 90%co fluency, which is optimal for cross-linking. In high co fluency conditions, cells tend to pile on each other leading to a decrease in the accessibility of the cell Permeable crosslinker to all cells. Place the plates back in the incubator until ready for crosslinking.
Prepare the DSP Crosslinker solution immediately before applying to the cells. DSP is highly hydrophobic and is therefore dissolved in DMSO to a concentration of 100 millimolar before diluting into the PBS calcium magnesium buffer. To further enhance the dilution of DSP into the PBS calcium magnesium buffer, warm and appropriate volume, add 10 microliters of the DSP and DMSO stock solution.
For every one milliliter of warm PBS calcium magnesium, add the D-S-P-D-M-S-O stock solution drop by drop with repeated mixing until all the DSP has dissolved. Also, prepare a vehicle control solution of 10 microliters of DMSO for every milliliter of PBS calcium magnesium. After diluting the DSP, keep all the PBS calcium magnesium solutions in an ice water bath for no longer than 10 minutes.
Next, prepare an ice water bath that will hold all the plates used in the experiment. Proceed to take the culture plates containing the cells from the 37 degree Celsius incubator and place them immediately into the ice water bath. Wash the cells twice with ice cold PBS calcium magnesium, using the same volumes prepared previously for the incubation with the crosslinker for six, well plate calculate two milliliters per well and so on.
After the second wash. Add either the vehicle control, which is the DMSO dissolved in buffer or the DSP crosslinking buffer solution to the cells incubate on ice for two hours and check approximately every 20 minutes to ensure that all cells are covered by solution. If some cells become exposed, tip and rotate the dish to cover them evenly again, it is normal for a small amount of DSP to precipitate.
During the two hour incubation with the cells before the two hour incubation has ended, prepare a one XDSP quenching solution by diluting the stalk into PBS calcium magnesium. Keep the solution on ice until using it to stop the cross-linking reaction by inactivating the DSP which has not reacted. When the two hours are up, remove the vehicle control and cross-linking solutions from the cells.
Add the ice cold DSP quenching solution and incubate on ice for 15 minutes. Meanwhile, dilute the complete protease inhibitor cocktail stock one to 50 into the cell lysis buffer. Once the 15 minutes are up, wash the cells two times with PBS calcium magnesium.
Then add the lysis buffer containing the protease inhibitors to the cells and rock at four degrees Celsius for 30 minutes. After 30 minutes of cell lysis, use a cell scraper to collect the cells from the plate. Be sure to keep the lysates on ice to minimize protease activity.
Spin the cell lysates for 15 minutes at four degrees Celsius in a benchtop mini centrifuge at top speed of 15, 000 Gs.Pipette the supernatant into a fresh tube and discard the pellet. Analyze protein levels using the Bradford assay and proceed with immunoprecipitation. Start preparing for the immunoprecipitation immediately after starting the two hour cross-linking reaction in screw top micro centrifuge tubes.
Combine 30 microliters of anti IgG immuno magnetic beads with 500 microliters of IP buffer. Add the antibody against the target of interest. The appropriate amount of antibody should be determined for each individual antigen.
In preliminary experiments, possible negative controls for the beads include no antibody, beads and isotype control antibody beads to bind the IP antibody to the magnetic beads. Incubate in an endover end rocker for two hours at room temperature At the end of the incubation, quick spin the tubes and slide them into the magnetic holder. The beads will concentrate at the side of the magnet.
Remove the solution containing unbound antibodies and immediately wash the beads to prevent them from drying out. After two washes, add 500 microliters of one microgram per milliliter of DSP negative or DSP positive cell lysate to the appropriate tubes. A peptide competition negative control can be prepared at this stage by adding the immunogenic peptide at a concentration determined in preliminary experiments to the lysate bead mix.
Another possible negative control prepared at this stage is a buffer only or lysate free reaction prepared by adding 500 microliters of lysis buffer only to the beads. Gently resuspend the beads for the experimental and control reactions. Do not vortex as this will cause the beads to break incubate at four degrees with end over end rotation for two hours.
At the end of two hours, remove the lysate using the magnetic holder. Quick spin and wash the beads for a total of six times with one milliliter of IP buffer. Incubating each wash at four degrees Celsius for five minutes.
To elude the bound complexes, add one x gel sample buffer to the IP tube just above the bead pellet. To pool the beads at the bottom of the tube, gently tap the bottom of the tube to get all of the beads resuspended in the gel sample buffer. Alternatively, for immuno affinity isolation at 30 microliters of buffer a supplemented with the immunogenic peptide to the beads.
The required concentration of the immunogenic peptide is determined empirically. After testing a range of 10 to 200 micromolar, incubate the beads at four degrees for two hours, tap the tubes every 30 minutes. Use the magnetic holder to remove the 30 microliters containing the eluded complexes and save in a clean tube wash with IP buffer and save the beads.
Add one x gel sample buffer to the beads for both elution methods. Heat the immuno magnetic beads re suspended in gel sample buffer at 75 degrees Celsius for five minutes in addition to denaturing the proteins. This step will also cleve the DSP molecule allowing for resolving the individual proteins in the complex run.
The samples on an SDS page gel for western blotting. The emptied immuno magnetic beads can be repelled by centrifugation and removed from the sample with a magnetic holder prior to loading. If the immuno affinity EUA is free of IgG both by protein stain of SDS page or by immuno blots, it is suitable for mass spectrometry.
Here is an example of a western blot of material from cross-linked and non cross-linked cell lysate, followed by immuno magnetic precipitation using a monoclonal antibody against the AP three subunit. Delta cross-linking allows for the detection of interacting proteins not seen in non cross-linked samples during immuno magnetic isolations. Negative controls shown no specific protein pull down using antibody free and antibody isotype matched control using an IgG directed against the transferrin receptor for both non cross-linked and cross-linked samples.
In this example, affinity peptide elution was used to recover the proteins bound to the IP antibody. Notice that the immuno antibody is absent in the supernat fraction and remains in the beat fraction during peptide elution. We've just shown you how to isolate labile MultiPro complexes using the chemical crosslinker DSP, followed by immuno magnetic affinity purification.
When doing this procedure, it's important to keep cells solutions and lysates chilled to maintain labile interactions during cross-linking and to minimize protease activity. So that's it. Thanks for watching and good luck with your experiments.
